Bacterial host strain expressing recombinant DSBC

ABSTRACT

The present invention provides a recombinant gram-negative bacterial cell comprising an expression vector comprising a recombinant polynucleotide encoding DsbC and one or more polynucleotides encoding an antibody or an antigen-binding fragment thereof specifically binding to CD154.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of InternationalPatent Application No. PCT/EP2012/002945, filed Jul. 13, 2012, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/560,356, filed Nov. 16, 2011, the disclosures of which are herebyincorporated by reference in their entirety, including all figures,tables and nucleic acid sequences.

The invention relates to a recombinant bacterial host strain,particularly E. coli. The invention also relates to a method forproducing a protein of interest in such a cell.

BACKGROUND OF THE INVENTION

Bacterial cells, such as E. coli, are commonly used for producingrecombinant proteins that do not require glycosylation. There are manyadvantages to using bacterial cells, such as E. coli, for producingrecombinant proteins, particularly due to the versatile nature ofbacterial cells as host cells, allowing gene insertion via plasmids. E.coli has been used to produce many recombinant proteins including humaninsulin.

Despite the many advantages to using bacterial cells to producerecombinant proteins, there are still significant limitations, includingpoor cell health phenotype.

Accordingly, there is still a need to provide new bacterial strainswhich provide advantageous means for producing recombinant proteins.

The generation of humoral and cell-mediated immunity is orchestrated bythe interaction of activated helper T cells with antigen-presentingcells (“APCs”) and effector T cells. Activation of the helper T cells isnot only dependent on the interaction of the antigen-specific T-cellreceptor (“TCR”) with its cognate peptide-MHC ligand, but also requirescoordinate binding and activation by a number of cell adhesion andcostimulatory molecules.

The natural receptor binding to CD40 is CD40 ligand (CD40-L=CD154), acritical costimulatory molecule that is expressed on the surface of CD4+T cells in an activation-dependent, temporally-restricted manner. CD154is also expressed, following activation, on a subset of CD8+ T cells,basophils, mast cells, eosinophils, natural killer cells, B cells,macrophages, dendritic cells and platelets. CD40 is constitutively andwidely expressed on the surface of many cell types, including B cellsand other antigen-presenting cells.

Signaling through CD40 after engagement with CD154 initiates a cascadeof cellular events that results in the activation of the CD40receptor-bearing cells and optimal CD4+ T cell priming. Morespecifically, the binding of CD154 to CD40 promotes the differentiationof B cells into antibody-secreting cells and memory B cells.

The pivotal role of CD154 in regulating the function of both the humoraland cell-mediated immune response has provoked great interest in the useof inhibitors of this pathway for therapeutic immunomodulation. As such,anti-CD154 antibodies have been shown to be beneficial in a wide varietyof models of immune response to other therapeutic proteins or genetherapy, allergens, autoimmunity and transplantation (see, e.g., U.S.Pat. No. 5,474,771 and WO 2008/118356, which are incorporated herein byreference in their entirety).

There is a need in the art to efficiently and cost-effectively producehigh amounts of antibodies or antibody fragments interfering with theinteraction of CD40 and CD154 suitable for therapeutic applications.

SUMMARY OF THE INVENTION

The present invention provides a recombinant gram-negative bacterialcell comprising:

-   -   a) a recombinant polynucleotide encoding DsbC; and    -   b) one or more polynucleotides encoding an antibody or an        antigen-binding fragment thereof specifically binding to CD154.

More specifically the present invention provides a recombinantgram-negative bacterial cell, characterized in that the cell:

-   -   a) comprises a recombinant polynucleotide encoding DsbC;    -   b) has reduced Tsp protein activity compared to a wild-type        cell, and    -   c) has one or more polynucleotides encoding an antibody or an        antigen-binding fragment thereof specifically binding to CD154.

In one embodiment the cell comprises a wild-type spr gene or a mutatedspr gene, for example capable of suppressing the reduced activity Tspphenotype.

The gram-negative bacterial cell according to the present inventionshows advantageous growth and protein production phenotypes.

More specifically the present invention provides a recombinantgram-negative bacterial cell comprising a recombinant polynucleotideencoding a DsbC polypeptide comprising a histidine (His)-tag, preferablywherein the DsbC polypeptide comprises the sequencehis-his-his-his-his-his (6× histidine).

More specifically the present invention provides a recombinantgram-negative bacterial cell comprising a recombinant polynucleotidecomprising a polynucleotide with the sequence according to SEQ ID NO: 45or SEQ ID NO: 51.

The present invention also provides an expression vector comprising arecombinant polynucleotide encoding DsbC and an antibody or anantigen-binding fragment thereof specifically binding to CD154.

More specifically the present invention provides an expression vectorcomprising a recombinant polynucleotide encoding a DsbC polypeptidecomprising a histidine (His)-tag, preferably wherein the DsbCpolypeptide comprises the sequence his-his-his-his-his-his (6×histidine).

More specifically the present invention provides an expression vectorcomprising a recombinant polynucleotide comprising a polynucleotide withthe sequence according to SEQ ID NO: 45 or SEQ ID NO: 51.

The present invention also provides a method for producing an antibodyor an antigen-binding fragment thereof specifically binding to CD154comprising:

-   -   a) culturing a recombinant gram-negative bacterial cell as        defined above in a culture medium under conditions effective to        express the antibody or the antigen-binding fragment thereof        specifically binding to CD154 and the recombinant polynucleotide        encoding DsbC; and    -   b) recovering the antibody or an antigen-binding fragment        thereof specifically binding to CD154 from the periplasm of the        recombinant gram-negative bacterial cell and/or the culture        medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of a vector for use in producing a cellaccording to an embodiment of the present invention.

FIG. 2 shows the structure of a compound comprising a modified Fab′fragment covalently linked via a cysteine residue to a lysyl-maleimidelinker, wherein each amino group on the lysyl residue has covalentlyattached to it a methoxy PEG residue, wherein n is between about 420 to450.

FIG. 3 shows the growth profile of anti-CD154 Fab′-expressing strainW3110 and the growth profile of anti-CD154 Fab′ and recombinantDsbC-expressing strain MXE016 (W3110 ΔTsp, spr C94A). It can be seenthat the MXE016 strain expressing recombinant DsbC exhibits improvedgrowth profile and growth rate in the initial batch phase compared tothe W3110 strain.

FIG. 4 shows total Fab′ yield (g/L) from the periplasm (closed symbols)and supernatant (open symbols) from the MXE016 strain expressingrecombinant DsbC compared to control strain W3110. The DsbC-expressingstrain shows higher periplasmic Fab′ expression compared to W3110.Further, the MXE016 strain expressing DsbC shows equivalentextracellular Fab′ levels compared to strain W3110.

FIG. 5 shows the results of a reverse phase HPLC analysis offermentation extractions. The wild-type strain W3110 expressinganti-CD154 Fab′ exhibits a high level of degraded Kappa light chains(light chain [LC] fragments). In contrast, strain MXE016 (W3110 ΔTsp,spr C94A) expressing recombinant DsbC and anti-CD154 Fab′ exhibitshardly any light chain fragments due to the absence of Tsp proteaseactivity.

FIG. 6 shows the harvest of anti-CD154 Fab′ (g/L) from fermentations instrain W3110 and in strain MXE016 (W3110 ΔTsp, spr C94A) expressingrecombinant DsbC. The harvest from strain MXE016 (W3110 ΔTsp, spr C94A)expressing recombinant DsbC is substantially higher and exhibitssubstantially less extracellular Fab′, which is beneficial asextracellular Fab′ is a marker of cell lysis risk and the extracellularFab is not easily recovered using the same process as used forperiplasmic Fab′.

FIG. 7 shows the viability of (a) strain W3110 cells and (b) strainMXE016 (W3110 ΔTsp, spr C94A) cells expressing recombinant DsbC; in bothcases (a) and (b) express anti-CD154 Fab′ (g/L). The strain MXE016 cells(W3110 ΔTsp, spr C94A) expressing recombinant DsbC exhibit a higherviability.

FIG. 8 shows total Fab′ yield (g/L) from fermentations of MXE016 strainsexpressing anti-CD154 Fab′. The right bar represents an MXE016 strainexpressing additionally recombinant DsbC. The MXE016 strain expressingrecombinant DsbC exhibits a higher yield.

FIG. 9 shows polynucleotide and amino acid sequences of a region withinthe ptr gene that was mutated.

FIG. 10 shows polynucleotide and amino acid sequences of a region withinthe Tsp gene that was mutated.

FIG. 11 shows polynucleotide and amino acid sequences of a region withinthe DegP gene that was mutated.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of CDRH1 of an anti-CD154antibody.

SEQ ID NO: 2 shows the amino acid sequence of CDRH2 of an anti-CD154antibody.

SEQ ID NO: 3 shows the amino acid sequence of CDRH3 of an anti-CD154antibody.

SEQ ID NO: 4 shows the amino acid sequence of CDRL1 of an anti-CD154antibody.

SEQ ID NO: 5 shows the amino acid sequence of CDRL2 of an anti-CD154antibody.

SEQ ID NO: 6 shows the amino acid sequence of CDRL3 of an anti-CD154antibody.

SEQ ID NO: 7 shows the polynucleotide and amino acid sequence of thevariable light chain (gL4) of an anti-CD154 antibody (342).

SEQ ID NO: 8 shows the amino acid sequence of the variable light chain(gL4) of an anti-CD154 antibody (342).

SEQ ID NO: 9 shows the polynucleotide and amino acid sequence of thevariable heavy chain (gH1) of an anti-CD154 antibody (342).

SEQ ID NO: 10 shows the amino acid sequence of the variable heavy chain(gH1) of an anti-CD154 antibody (342).

SEQ ID NO: 11 shows the polynucleotide and amino acid sequencecomprising the variable and constant region of the light chain (gL4) ofan anti-CD154 antibody fragment.

SEQ ID NO: 12 shows the amino acid sequence comprising the variable andconstant region of the light chain (gL4) of an anti-CD154 antibodyfragment.

SEQ ID NO: 13 shows the polynucleotide and amino acid sequence of aheavy chain fragment of an anti-CD154 antibody comprising the variableand the CH1 region with deletions in the hinge region.

SEQ ID NO: 14 shows the amino acid sequence of a heavy chain fragment ofan anti-CD154 antibody comprising the variable and the CH1 region withdeletions in the hinge region.

SEQ ID NO: 15 shows the polynucleotide and amino acid sequence of aheavy chain fragment of an anti-CD154 antibody comprising the variable,the CH1 and the hinge region.

SEQ ID NO: 16 shows the amino acid sequence of a heavy chain fragment ofan anti-CD154 antibody comprising the variable, the CH1 and the hingeregion.

SEQ ID NO: 17 shows the polynucleotide and amino acid sequence of akappa light chain of an anti-CD154 antibody (342) including the signalpeptide (amino acids 1-22).

SEQ ID NO: 18 shows the amino acid sequence of a kappa light chain of ananti-CD154 antibody (342) including the signal peptide (amino acids1-22).

SEQ ID NO: 19 shows the polynucleotide and amino acid sequence of theentire heavy chain of an aglycosylated IgG₄ anti-CD154 antibody.

SEQ ID NO: 20 shows the amino acid sequence of the entire heavy chain ofan aglycosylated IgG₄ anti-CD154 antibody.

SEQ ID NO: 21 shows the polynucleotide sequence coding for gL4 and gH1(no hinge).

SEQ ID NO: 22 shows the polynucleotide sequence coding for gL4 and gH1.

SEQ ID NO: 23 shows the polynucleotide and amino acid sequence forwild-type E. coli spr (GenBank accession no. D86610).

SEQ ID NO: 24 shows the amino acid sequence for wild-type E. coli spr(GenBank accession no. D86610).

SEQ ID NO: 25 shows the polynucleotide and amino acid sequence forwild-type E. coli Tsp with the signal sequence (GenBank accession no.M75634).

SEQ ID NO: 26 shows the amino acid sequence for wild-type E. coli Tspwith the signal peptide (GenBank accession no. M75634).

SEQ ID NO: 27 shows the amino acid sequence for wild-type E. coli DsbC(NCBI Reference Sequence AP_003452).

SEQ ID NO: 28 shows the polynucleotide sequence for knockout mutated Tspgene.

SEQ ID NO: 29 shows the polynucleotide and amino acid sequence forwild-type E. coli DegP.

SEQ ID NO: 30 shows the amino acid sequence for wild-type E. coli DegP.

SEQ ID NO: 31 shows the polynucleotide and amino acid sequence for DegPcomprising the point mutation S210A and an Ase I restriction marker.

SEQ ID NO: 32 shows the amino acid sequence for DegP comprising thepoint mutation S210A and an Ase I restriction marker.

SEQ ID NO: 33 to 36 show dicistronic intergenic sequences (IGS) IGS1,IGS2, IGS3 and IGS4, respectively.

SEQ ID NO: 37 shows the polynucleotide and amino acid sequence forwild-type E. coli OmpT.

SEQ ID NO: 38 shows the amino acid sequence for wild-type E. coli OmpT.

SEQ ID NO: 39 shows the polynucleotide and amino acid sequence forknockout mutated E. coli OmpT.

SEQ ID NO: 40 shows the amino acid sequence for knockout mutated E. coliOmpT.

SEQ ID NO: 41 shows the polynucleotide and amino acid sequence of themutated E. coli OmpT comprising the point mutations D210A and H212A.

SEQ ID NO: 42 shows the amino acid sequence of the E. coli OmpTcomprising the point mutations D210A and H212A.

SEQ ID NO: 43 shows the polynucleotide and amino acid sequence ofwild-type E. coli DsbC.

SEQ ID NO: 44 shows the amino acid sequence of wild-type E. coli DsbC.

SEQ ID NO: 45 shows the polynucleotide and amino acid sequence of E.coli DsbC lacking an EcoRI restriction site with a His-tag.

SEQ ID NO: 46 shows the amino acid sequence of E. coli DsbC lacking anEcoRI restriction site with a His-tag.

SEQ ID NO: 47 shows the a polynucleotide sequence of primer 6283 Tsp 3′.

SEQ ID NO: 48 shows the a polynucleotide sequence of primer 6283 Tsp 5′.

SEQ ID NO: 49 shows the polynucleotide and amino acid sequence ofwild-type E. coli ptr (protease III according to GenBank accessionnumber X06227).

SEQ ID NO: 50 shows the amino acid sequence of wild-type E. coli ptr(protease III according to GenBank accession number X06227).

SEQ ID NO: 51 shows the polynucleotide and amino acid sequence ofwild-type E. coli DsbC with a His-tag.

SEQ ID NO: 52 shows the amino acid sequence of wild-type E. coli DsbCwith a His-tag.

DETAILED DESCRIPTION OF THE INVENTION

Tsp (also known as Prc) is a 60 kDa periplasmic protease. The firstknown substrate of Tsp was Penicillin-binding protein-3 (PBP3)(Determination of the cleavage site involved in C-terminal processing ofpenicillin-binding protein 3 of Escherichia coli (Hara et al. 4799-813;Nagasawa et al. 5890-93)) but it was later discovered that the Tsp wasalso able to cleave phage tail proteins and, therefore, it was renamedas Tail Specific Protease (Tsp). Silber, Keiler, and Sauer (295-99) andSilber and Sauer (237-40) describe a prc deletion strain (KS1000)wherein the mutation was created by replacing a segment of the prc genewith a fragment comprising a Kan^(r) marker.

The reduction of Tsp (prc) activity is desirable to reduce theproteolysis of proteins of interest. Fab proteolysis may manifest itselfas the presence of impurities such as a fragment which can be referredto as the light chain impurity.

However, it was found that cells lacking protease prc showthermosensitive growth at low osmolarity. Hara et al. isolatedthermoresistant revertants containing extragenic suppressor (spr)mutations (Hara et al. 63-72). Spr is an 18 kDa membrane-boundperiplasmic protease and the substrates of spr are Tsp andpeptidoglycans in the outer membrane involved in cell wall hydrolysisduring cell division. The spr gene is designated as UniProtKB/Swiss-ProtP0AFV4 (SPR_ECOLI).

Protein disulfide isomerase is an enzyme that catalyzes the formationand breakage of disulfide bonds between cysteine residues withinproteins as they fold. It is known to co-express proteins which catalyzethe formation of disulfide bonds to improve protein expression in a hostcell. WO 98/56930 discloses a method for producing heterologousdisulfide bond-containing polypeptides in bacterial cells wherein aprokaryotic disulfide isomerase, such as DsbC or DsbG, is co-expressedwith a eukaryotic polypeptide. U.S. Pat. No. 6,673,569 discloses anartificial operon comprising polynucleotides encoding each of DsbA,DsbB, DsbC and DsbD for use in producing a foreign protein. EP0786009discloses a process for producing a heterologous polypeptide in bacteriawherein the expression of nucleic acid encoding DsbA or DsbC is inducedprior to the induction of expression of nucleic acid encoding theheterologous polypeptide.

DsbC is a prokaryotic protein found in the periplasm of E. coli whichcatalyzes the formation of disulfide bonds in E. coli. DsbC has an aminoacid sequence length of 236 (including the signal peptide) and amolecular weight of 25.6 KDa (UniProt No. P0AEG6). DsbC was firstidentified in 1994 (Missiakas, Georgopoulos, and Raina 2013-20;Shevchik, Condemine, and Robert-Baudouy 2007-12).

It has been surprisingly found that the over-expression of DsbC in agram-negative bacterial cell reduces lysis during cultivation of cellslacking protease Tsp. Accordingly, the present inventors have provided anew strain having advantageous properties for producing a protein ofinterest.

The gram-negative bacterial cell having the above specific combinationof genetic modifications shows advantageous growth and proteinproduction phenotypes.

In one embodiment the cell's genome is preferably isogenic to awild-type bacterial cell except for the modification required to reduceTsp protein activity compared to a wild-type cell.

In a further embodiment the cell according to the present invention hasreduced Tsp protein activity compared to a wild-type cell and comprisesa recombinant polynucleotide encoding DsbC and an altered spr protein.In this embodiment the cell's genome is preferably isogenic to awild-type bacterial cell except for the mutated spr gene and amodification leading to reduced or absent expression of the Tsp proteincompared to a wild-type cell.

The terms “protein” and “polypeptide” are used interchangeably herein,unless the context indicates otherwise. “Peptide” is intended to referto 20 or fewer amino acids.

The term “polynucleotide” includes a gene, DNA, cDNA, RNA, mRNA, andanalogues thereof, including, but not limited to, locked nucleic acid(LNA), peptide nucleic acid (PNA), morpholino nucleic acid, glycolnucleic acid (GNA), threose nucleic acid (TNA), etc., unless the contextindicates otherwise.

As used herein, the term “comprising” in context of the presentspecification should be interpreted as “including”.

The non-mutated cell or control cell in the context of the presentinvention means a cell of the same type as the recombinant gram-negativecell of the invention wherein the cell has not been modified to carrythe recombinant polynucleotide encoding DsbC and one or morepolynucleotides encoding an antibody or an antigen-binding fragmentthereof specifically binding to CD154. For example, a non-mutated cellmay be a wild-type cell and may be derived from the same population ofhost cells as the cells of the invention before modification tointroduce any recombinant polynucleotide(s).

The expressions “cell”, “cell line”, “cell culture” and “strain” areused interchangeably.

The term “isogenic” in the context of the present invention means thatthe cell comprises the same or substantially the same nucleic acidsequence(s) compared to a wild-type cell except for the elementsincorporated therein that characterize the invention, for example therecombinant polynucleotide encoding DsbC and the one or morepolynucleotides encoding an antibody or an antigen-binding fragmentthereof specifically binding to CD154, and optionally a modificationleading to reduced or absent expression of the Tsp protein, andoptionally a mutated spr gene. In this embodiment the cell according tothe present invention comprises no further non-naturally occurring orgenetically engineered changes to its genome.

In one embodiment wherein the polynucleotide encoding DsbC and/or theone or more polynucleotides encoding the antibody or antigen-bindingfragment thereof are inserted into the cell's genome, the cell accordingto the present invention may have substantially the same genomicsequence compared to the wild-type cell except for the polynucleotideencoding DsbC and/or the one or more polynucleotides encoding theantibody or antigen-binding fragment thereof, and optionally amodification resulting in a reduced or absent expression of the Tspprotein or the expression of a Tsp protein with reduced proteaseactivity, and optionally a mutated spr gene coding for a protein withreduced activity as compared to the wild-type, taking into account anynaturally occurring mutations which may occur. In one embodiment,wherein the polynucleotide encoding DsbC and/or the one or morepolynucleotides encoding the antibody or antigen-binding fragmentthereof are inserted into the cell's genome, the cell according to thepresent invention may have exactly the same genomic sequence compared tothe wild-type cell except for the polynucleotide encoding DsbC and/orthe one or more polynucleotides encoding the antibody or antigen-bindingfragment thereof.

The polynucleotide encoding DsbC may be present on a suitable expressionvector transformed into the cell and/or integrated into the host cell'sgenome. In the embodiment where the polynucleotide encoding DsbC isinserted into the host cell's genome, the cell of the present inventiondiffers from a wild-type cell due to the inserted polynucleotideencoding the DsbC. In this embodiment, the host cell's genome may beisogenic compared to a wild-type cell genome except for the recombinantpolynucleotide encoding DsbC.

Preferably the polynucleotide encoding DsbC is in an expression vectorin the cell, thereby causing minimal disruption to the host cell'sgenome.

The one or more polynucleotides encoding the antibody or anantigen-binding fragment thereof specifically binding to CD154 may becontained within a suitable expression vector transformed into the celland/or integrated into the host cell's genome. In the embodiment wherethe polynucleotide encoding the antibody or antigen-binding fragmentthereof specifically binding to CD154 is inserted into the host'sgenome, the cell of the present invention differs from a wild-type celldue to the inserted polynucleotide(s) encoding the antibody orantigen-binding fragment thereof. In this embodiment, the host cell'sgenome may be isogenic compared to a wild-type cell genome except forthe polynucleotide(s) encoding the antibody or antigen-binding fragmentthereof. Preferably the polynucleotide encoding the protein of interestis in an expression vector in the cell, thereby causing minimaldisruption to the host cell's genome.

In one embodiment the recombinant polynucleotide encoding DsbC and thepolynucleotide encoding the antibody or antigen-binding fragment thereofspecifically binding to CD154 are inserted into the host's genome. Inthis embodiment, the cell of the present invention differs from awild-type cell due to the inserted recombinant polynucleotide encodingDsbC and the one or more polynucleotide encoding the antibody orantigen-binding fragment thereof, and optionally a modificationresulting in a reduced or absent expression of the Tsp protein or theexpression of a Tsp protein with reduced protease activity, andoptionally a mutated spr gene coding for a protein with reduced activityas compared to the wild-type. In this embodiment, the host cell's genomemay be isogenic compared to a wild-type cell genome except for therecombinant polynucleotide encoding DsbC and the one or morepolynucleotides encoding the antibody or antigen-binding fragmentthereof.

In a preferred embodiment the recombinant polynucleotide encoding DsbCand the polynucleotide encoding the antibody or antigen-binding fragmentthereof specifically binding to CD154 are present in the same ordifferent expression vectors in the cell, thereby causing minimaldisruption to the host cell's genome. In this embodiment the cell'sgenome may be substantially the same or exactly the same compared to thegenome of a wild-type cell.

In one embodiment there is provided a recombinant E. coli cell that hasreduced Tsp activity and optionally an spr gene or a mutant thereof,wherein modifications to the Tsp activity and any mutation in the sprgene are effected through changes in the cell's genome. The cellaccording to this embodiment may be transformed with a vector such as aplasmid encoding DsbC and the antibody or a binding fragment thereofspecifically binding to CD154. In one embodiment the vector or plasmidis not integrated into the genome of the cell.

The term “wild-type” in the context of the present invention means astrain of a gram-negative bacterial cell as it may occur in nature ormay be isolated from the environment, which does not carry anyrecombinant polynucleotide or genetically engineered mutations. Anexample of a wild-type strain of E. coli is the K-12 strain and itspedigree strain W3110. E. coli strain K-12 has been in cultivation for90 years now (Bachmann 525-57). E. coli strain K-12 and its pedigreestrains such as W3110 are well-known in the art. Strain W3110 isavailable for example from the American Tissue Culture Collection (ATCC)under catalog no. 27325. W3110 has the genotype: F⁻, λ⁻, IN(rrnD-rrnE)1,rph-1.

The present inventors have provided a recombinant gram-negativebacterial cell suitable for expressing an antibody or an antigen-bindingfragment thereof specifically binding to CD154 which comprises arecombinant polynucleotide encoding DsbC.

The cells according to the present invention comprise a recombinantpolynucleotide encoding DsbC. As used herein, a “recombinantpolypeptide” refers to a protein that is constructed or produced usingrecombinant DNA technology. The polynucleotide encoding DsbC may beidentical to the endogenous polynucleotide encoding DsbC found inwild-type bacterial cells. Alternatively, the recombinant polynucleotideencoding DsbC is a mutated version of the wild-type DsbC polynucleotide,for example being altered such that the restriction site, such as anEcoRI site, is removed from the DsbC protein and/or the his-tag. Anexample of a modified DsbC polynucleotide for use in the presentinvention is shown in SEQ ID NO: 45, which encodes the his-tagged DsbCsequence shown in SEQ ID NO: 46.

DsbC is characterized by the presence of an active site comprising aminoacids -CXXC- wherein XX represents the amino acids GY. Variants of DsbCinclude wherein each X represents an amino acid independently selected(with the proviso that XX does not represent GY). Examples of XX includeNY, SF, TF, MF, GF, HH, VH, SH, RF, FA, GA, MA, GI or AV.

In one embodiment the host cell of the invention comprises a variant ofDsbC, for example where the active site is altered, in particular asdescribed above.

In one embodiment the variant of DsbC has at least the biologicalactivity of the wild-type protein, for example as measured in an invitro assay.

In one embodiment the variant of DsbC has a greater biological activitythan the wild-type protein, for example as measured in an in vitroassay.

In one embodiment the variant of DsbC has an alteration in the activesite -CXXC- wherein XX represents NY, SF, TF, MF, GF, HH, VH, or SH.

In one embodiment the DsbC is wild-type.

The present inventors have identified that the selection of theexpression of recombinant polynucleotide encoding DsbC in a bacterialcell provides an improved host cell for expressing an antibody or anantigen-binding fragment thereof specifically binding to CD154. Thecells provided by the present invention have improved cell health andgrowth phenotype compared to wild-type bacterial cells.

Improved cell health as employed herein is intended to refer to one ormore improved properties in comparison to cells which do not carry thefeatures according to the present invention, for example a lowerpropensity for cell lysis at the growth phase or after induction ofexpression of a heterologous protein in the cell or other beneficialproperty as known to the skilled artisan.

The cells according to the present invention exhibit improved protein,such as antibody or antibody fragment, production yield compared towild-type bacterial cells. The improved protein yield may be the rate ofprotein production and/or the duration of protein production from thecell. The improved protein yield may be the periplasmic protein yieldand/or the supernatant protein yield. The recombinant bacterial cellsmay be capable of a faster rate of production of the protein, such as anantibody or fragment thereof, and, therefore, the same quantity of aprotein of interest may be produced in a shorter time compared to anon-mutated bacterial cell. The faster rate of production of a proteinof interest may be especially significant over the initial period ofgrowth of the cell, for example over the first 5, 10, 20 or 30 hourspost induction of protein expression.

The cells according to the present invention preferably express a yieldin the periplasm and/or media of approximately 1.0 g/L, 1.5 g/L, 1.8g/L, 2.0 g/L, 2.4 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L or 4.0 g/L of theantibody.

Advantageously the reduced Tsp protein activity and/or the co-expressionof DsbC leads to reduced generation of the undesirable impurity referredto herein as the light chain fragment (LC), see for example FIG. 5.

The skilled person would easily be able to test a candidate cell cloneto see if it has the desired yield of a protein of interest usingmethods well-known in the art, including a fermentation method, ELISAand protein G HPLC. Suitable fermentation methods are described inHumphreys et al. (193-202) and Backlund et al. (358-65), which areincorporated herein by reference in their entirety. The skilled personwould also easily be able to test secreted protein to see if the proteinis correctly folded using methods well-known in the art, such as proteinG HPLC, circular dichroism, NMR, x-ray crystallography and epitopeaffinity measurement methods.

In one embodiment the cell according to the present invention alsoexpresses or overexpresses, as compared to the corresponding wild-typecell (such as the E. coli W3110 K-12 strain), one or more furtherproteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD;    -   one or more proteins capable of facilitating protein secretion        or translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulfide bond        formation, such as DsbA, DsbB, DsbD, and DsbG.

One of more of the above proteins may be integrated into the cell'sgenome and/or inserted in an expression vector.

In one embodiment the cell according to the present invention does notexpress or expresses at a level which is at least 50%, 75% or 90% lowerthan the level of the corresponding wild-type cell (such as the E. coliW3110 K-12 strain) one or more of the following further proteins:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD;    -   one or more proteins capable of facilitating protein secretion        or translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and    -   one or more proteins capable of facilitating disulfide bond        formation, such as DsbA, DsbB, DsbD, and DsbG.

In one embodiment the cell according to the present invention alsoexpresses one or more further proteins selected from FkpA, Skp and acombination thereof.

In one embodiment the cell further comprises one or more of thefollowing mutated genes:

-   -   a) a mutated spr gene;    -   b) a mutated Tsp gene, wherein the mutated Tsp gene encodes a        Tsp protein having reduced protease activity or is a knockout        mutated Tsp gene;    -   c) a mutated DegP gene encoding a DegP protein having chaperone        activity and reduced protease activity;    -   d) a mutated ptr gene, wherein the mutated ptr gene encodes a        Protease III protein having reduced protease activity or is a        knockout mutated ptr gene; and    -   e) a mutated OmpT gene, wherein the mutated OmpT gene encodes an        OmpT protein having reduced protease activity, for example as        shown in SEQ ID NO: 42, or is a knockout mutated OmpT gene, for        example as shown in SEQ ID NO: 40.

In a basic embodiment of the invention the gram-negative bacterial celldoes not carry a knockout mutated Tsp gene, such as being deficient inchromosomal Tsp.

The latter mutation is particular important in production of antibodiesand fragments thereof specifically binding to CD154 because Tsp proteaseactivity may result in cleavage of the antibody product in the elbowregions, thereby generating a by-product in significant quantities andreducing yield of the desired product.

Thus in one embodiment the cell according to the present inventioncomprises a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tspprotein having reduced protease activity or is a knockout mutated Tspgene and also encodes a DsbC protein in addition to an antibody orfragment specific to CD154.

In embodiments of the present invention the cell further comprises amutated spr gene. The spr protein is an E. coli membrane-boundperiplasmic protease.

The wild-type amino acid sequence of the Spr protein is shown in SEQ IDNO: 24 with the signal sequence at the N-terminus (amino acids 1-26according to UniProt Accession Number P0AFV4). The amino acid numberingof the Spr protein sequence in the present invention includes the signalsequence. Accordingly, the amino acid 1 of the Spr protein is the firstamino acid (Met) shown in SEQ ID NO: 24.

In the embodiments wherein the cell according to the present inventioncomprises a mutated spr gene, the mutated spr gene is preferably thecell's chromosomal spr gene. The mutated spr gene encodes an Spr proteincapable of suppressing a disadvantageous phenotype associated with acell comprising a mutated Tsp gene. Cells carrying a mutated Tsp genemay have a good cell growth rate but one limitation of these cells istheir tendency to lyse, especially at high cell densities. Accordinglythe phenotype of a cell comprising a mutated Tsp gene is a tendency tolyse, especially at high cell densities.

Cells carrying a mutated Tsp gene show thermosensitive growth at lowosmolarity. However, the spr mutations carried by the cells of thepresent invention, when introduced into a cell having reduced Tspactivity, suppress this phenotype of thermosensitive growth at lowosmolarity and the cell exhibits reduced lysis, particularly at highcell densities. This “thermosensitive growth” phenotype of a cell may beeasily measured by a person skilled in the art during the shake flask orhigh cell density fermentation technique. The suppression of the celllysis is apparent from the improved growth rate and/or recombinantprotein production, particularly in the periplasm, exhibited by a cellcarrying the spr mutant and having reduced Tsp activity compared to acell carrying the Tsp mutant and a wild-type spr.

The cells according to the present invention preferably comprise amutant spr gene encoding an spr protein having a change of one or moreamino acids selected from N31, R62, I70, Q73, C94, S95, V98, Q99, R100,L108, Y115, D133, V135, L136, G140, R144, H145, G147, H157 and W174,more preferably at one or more amino acids selected from C94, S95, V98,Y115, D133, V135, H145, G147, H157 and W174. Preferably the mutant sprgene encodes a spr protein having a change of one or more amino acidsselected from N31, R62, I70, Q73, C94, S95, V98, Q99, R100, L108, Y115,D133, V135, L136, G140, R144, H145, G147 and H157, more preferably atone or more amino acids selected from C94, S95, V98, Y115, D133, V135,H145, G147 and H157. In this embodiment, the spr protein preferably doesnot have any further amino acid changes. Preferably, the mutant spr geneencodes an spr protein having a change of one or more amino acidsselected from N31, R62, I70, Q73, S95, V98, Q99, R100, L108, Y115, D133,V135, L136, G140, R144 and G147, more preferably at one or more aminoacids selected from S95, V98, Y115, D133, V135 and G147. In thisembodiment, the spr protein preferably does not have any further aminoacid changes.

The present inventors have identified spr changes which are capable ofsuppressing the growth phenotype of a cell comprising a mutated Tspgene.

The inventors have also surprisingly found that cells carrying arecombinant DsbC gene and a mutated spr gene and having reduced Tspprotein activity compared to a wild-type cell exhibit increased cellgrowth rate and increased cell survival duration compared to a cellcomprising a mutated Tsp gene. Specifically, cells carrying arecombinant DsbC gene and a change in the spr protein and having reducedTsp protein activity exhibit reduced cell lysis during cultivationcompared to cells carrying a mutated Tsp gene.

The change of one or more of the above spr amino acids may be the resultof any suitable missense mutation to one, two or three of thenucleotides encoding the amino acid. The mutation changes the amino acidresidue to any suitable amino acid which results in a mutated sprprotein capable of suppressing the phenotype of a cell comprising amutated Tsp gene. The missense mutation may change the amino acid to onewhich is a different size and/or has different chemical propertiescompared to the wild-type amino acid.

In one embodiment the change is with respect to one, two or three of thecatalytic triad of amino acid residues of C94, H145, and H157 (Araminiet al. 9715-17).

Accordingly, the mutated spr gene may comprise:

-   -   a mutation affecting the amino acid C94;    -   a mutation affecting the amino acid H145;    -   a mutation affecting the amino acid H157;    -   a mutation affecting the amino acids C94 and H145;    -   a mutation affecting the amino acids C94 and H157;    -   a mutation affecting the amino acids H145 and H157; or    -   a mutation affecting the amino acids C94, H145 and H157.

In this embodiment, the spr protein preferably does not have any furtheramino acid changes.

One, two or three of C94, H145 and H157 may be changed to any suitableamino acid which results in an spr protein capable of suppressing thephenotype of a cell comprising a mutated Tsp gene. For example, one, twoor three of C94, H145, and H157 may be changed to a small amino acidsuch as Gly or Ala. Accordingly, the spr protein may have one, two orthree of the mutations resulting in C94A (i.e., cysteine at position 94changed to alanine), H145A (i.e., histidine at position 145 changed toalanine) and H157A (i.e., histidine at position 157 changed to alanine).Preferably, the spr gene comprises the missense mutation leading toH145A, which has been found to produce an spr protein capable ofsuppressing the phenotype of a cell comprising a mutated Tsp gene.

The designation for a substitution mutant herein consists of a letterfollowed by a number followed by a letter. The first letter designatesthe amino acid in the wild-type protein, the number refers to the aminoacid position where the amino acid substitution is being made, and thesecond letter designates the amino acid that is used to replace thewild-type amino acid.

In a preferred embodiment the mutant spr protein comprises a change ofone or more amino acids selected from N31, R62, I70, Q73, S95, V98, Q99,R100, L108, Y115, D133, V135, L136, G140, R144 and G147, preferably achange of one or more amino acids selected from S95, V98, Y115, D133,V135 and G147. In this embodiment, the spr protein preferably does nothave any further mutations. Accordingly, the mutated spr gene maycomprise:

-   -   a mutation affecting the amino acid N31;    -   a mutation affecting the amino acid R62;    -   a mutation affecting the amino acid 170;    -   a mutation affecting the amino acid Q73;    -   a mutation affecting the amino acid S95;    -   a mutation affecting the amino acid V98;    -   a mutation affecting the amino acid Q99;    -   a mutation affecting the amino acid R100;    -   a mutation affecting the amino acid L108;    -   a mutation affecting the amino acid Y115;    -   a mutation affecting the amino acid D133;    -   a mutation affecting the amino acid V135;    -   a mutation affecting the amino acid L136;    -   a mutation affecting the amino acid G140;    -   a mutation affecting the amino acid R144; or    -   a mutation affecting the amino acid G147.

In one embodiment the mutant spr gene comprises multiple mutationsaffecting the amino acids:

-   -   S95 and Y115;    -   N31, Q73, R100 and G140;    -   Q73, R100 and G140;    -   R100 and G140;    -   Q73 and G140;    -   Q73 and R100;    -   R62, Q99 and R144; or    -   Q99 and R144.

One or more of the amino acids N31, R62, I70, Q73, S95, V98, Q99, R100,L108, Y115, D133, V135, L136, G140, R144 and G147 may be changed to anysuitable amino acid which results in an spr protein capable ofsuppressing the phenotype of a cell comprising a mutated Tsp gene. Forexample, one or more of N31, R62, I70, Q73, S95, V98, Q99, R100, L108,Y115, D133, V135, L136, G140 and R144 may be changed to a small aminoacid such as Gly or Ala.

In a preferred embodiment the spr protein comprises one or more of thefollowing changes: N31Y, R62C, I70T, Q73R, S95F, V98E, Q99P, R100G,L108S, Y115F, D133A, V135D or V135G, L136P, G140C, R144C and G147C.Preferably the spr protein comprises one or more of the followingchanges: S95F, V98E, Y115F, D133A, V135D or V135G and G147C. In thisembodiment, the spr protein preferably does not have any further aminoacid changes.

In one embodiment the spr protein has only one amino acid changeselected from N31Y, R62C, I70T, Q73R, C94A, S95F, V98E, Q99P, R100G,L108S, Y115F, D133A, V135D or V135G, L136P, G140C, R144C and G147C, inparticular C94A. In this embodiment, the spr protein preferably does nothave any further amino acid changes.

In a further embodiment the spr protein has multiple changes selectedfrom:

-   -   S95F and Y115F;    -   N31Y, Q73R, R100G and G140C;    -   Q73R, R100G and G140C;    -   R100G and G140C;    -   Q73R and G140C;    -   Q73R and R100G;    -   R62C, Q99P and R144C; or    -   Q99P and R144C.

Preferably, the mutant spr gene encodes an spr protein having amino acidchanges selected from C94A, D133A, H145A and H157A, in particular C94A.

In a further embodiment the mutated spr gene encodes an spr proteinhaving the amino acid change W174R. In an alternative embodiment the sprprotein does not have the amino acid change W174R.

The cell according to the present invention has reduced Tsp proteinactivity compared to a wild-type cell. The expression “reduced Tspprotein activity compared to a wild-type cell” means that the Tspactivity of the cell is reduced compared to the Tsp activity of awild-type cell. The cell may be modified by any suitable means to reducethe activity of Tsp.

In one embodiment the reduced Tsp activity is from modification of theendogenous polynucleotide encoding Tsp and/or associated regulatoryexpression sequences. The modification may reduce or stop Tsp genetranscription and translation or may provide an expressed Tsp proteinhaving reduced protease activity compared to the wild-type Tsp protein.

In one embodiment an associated regulatory expression sequence ismodified to reduce Tsp expression. For example, the promoter for the Tspgene may be mutated to prevent expression of the gene.

In a preferred embodiment the cell according to the present inventioncarries a mutated Tsp gene encoding a Tsp protein having reducedprotease activity or a knockout mutated Tsp gene. Preferably thechromosomal Tsp gene is mutated.

As used herein, “Tsp gene” means a gene encoding protease Tsp (alsoknown as Prc) which is a periplasmic protease capable of acting onPenicillin-binding protein-3 (PBP3) and phage tail proteins. Thesequence of the wild-type Tsp gene is shown in SEQ ID NO: 25 and thesequence of the wild-type Tsp protein is shown in SEQ ID NO: 26.

Reference to the mutated Tsp gene or mutated Tsp gene encoding Tsprefers to either a mutated Tsp gene encoding a Tsp protein havingreduced protease activity or a knockout mutated Tsp gene, unlessotherwise indicated.

The expression “mutated Tsp gene encoding a Tsp protein having reducedprotease activity” in the context of the present invention means thatthe mutated Tsp gene does not have the full protease activity comparedto the wild-type non-mutated Tsp gene.

Preferably, the mutated Tsp gene encodes a Tsp protein having 50% orless, 40% or less, 30% or less, 20% or less, 10% or less or 5% or lessof the protease activity of a wild-type non-mutated Tsp protein. Morepreferably, the mutated Tsp gene encodes a Tsp protein having noprotease activity. In this embodiment the cell is not deficient inchromosomal Tsp, i.e., the Tsp gene sequence has not been deleted ormutated to prevent expression of any form of Tsp protein.

Any suitable mutation may be introduced into the Tsp gene in order toproduce a protein having reduced protease activity. The proteaseactivity of a Tsp protein expressed from a gram-negative bacterium maybe easily tested by a person skilled in the art by any suitable methodin the art, such as the method described in Keiler et al. (Keiler andSauer 28864-68), which is incorporated by reference herein in itsentirety, wherein the protease activity of Tsp was tested.

Tsp has been reported in Keiler et al. (supra) as having an active sitecomprising residues S430, D441 and K455 and residues G375, G376, E433and T452 are important for maintaining the structure of Tsp. Keiler etal. (supra) reports findings that the mutated Tsp genes leading to theamino acid changes S430A, D441A, K455A, K455H, K455R, G375A, G376A,E433A and T452A had no detectable protease activity. It is furtherreported that the mutated Tsp gene leading to S430C displayed about5-10% wild-type activity. Accordingly, the Tsp mutation to produce aprotein having reduced protease activity may comprise a mutation, suchas a missense mutation, leading to a change of one or more of residuesS430, D441, K455, G375, G376, E433 and T452. Preferably the Tsp mutationto produce a protein having reduced protease activity may comprise amutation, such as a missense mutation, affecting one, two or all threeof the active site residues S430, D441 and K455.

Accordingly the mutated Tsp gene may comprise:

-   -   a mutation affecting the amino acid S430;    -   a mutation affecting the amino acid D441;    -   a mutation affecting the amino acid K455;    -   a mutation affecting the amino acids S430 and D441;    -   a mutation affecting the amino acids S430 and K455;    -   a mutation affecting the amino acids D441 and K455; or    -   a mutation affecting the amino acids S430, D441 and K455.

One or more of residues S430, D441, K455, G375, G376, E433 and T452 maybe changed to any suitable amino acid which results in a protein havingreduced protease activity. Examples of suitable changes are S430A,S430C, D441A, K455A, K455H, K455R, G375A, G376A, E433A and T452A. Themutated Tsp gene may comprise one, two or three mutations leading tochanges to the active site residues; for example the gene may comprise:

-   -   S430A or S430C; and/or    -   D441A; and/or    -   K455A or K455H or K455R.

Preferably, the Tsp gene has the mutation leading to S430A or S430C.

The expression “knockout mutated Tsp gene” in the context of the presentinvention means that the Tsp gene comprises one or more mutations whichprevent expression of the Tsp protein encoded by the wild-type gene toprovide a cell deficient in Tsp protein. The knockout gene may bepartially or completely transcribed but not translated into the encodedprotein. The knockout mutated Tsp gene may be mutated in any suitableway, for example by one or more deletion, insertion, point, missense,nonsense and frameshift mutations, to cause no expression of theprotein. For example, the gene may be knocked out by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence.

In a preferred embodiment the Tsp gene is not mutated by insertion of aforeign DNA sequence, such as an antibiotic resistance marker, into thegene coding sequence. In one embodiment the Tsp gene comprises amutation to the gene start codon and/or one or more stop codonspositioned downstream of the gene start codon and upstream of the genestop codon, thereby preventing expression of the Tsp protein. Themutation to the start codon may be a missense mutation of one, two orall three of the nucleotides of the start codon. Alternatively oradditionally the start codon may be mutated by an insertion or deletionframeshift mutation. The Tsp gene comprises two ATG codons at the 5′ endof the coding sequence; one or both of the ATG codons may be mutated bya missense mutation. The Tsp gene may be mutated at the second ATG codon(codon 3) to TCG, as shown in FIG. 10. The Tsp gene may alternatively oradditionally comprise one or more stop codons positioned downstream ofthe gene start codon and upstream of the gene stop codon. Preferably theknockout mutated Tsp gene comprises both a missense mutation to thestart codon and one or more inserted stop codons. In a preferredembodiment the Tsp gene is mutated to delete “T” from the fifth codon,thereby causing a frameshift resulting in stop codons at codons 11 and16, as shown in FIG. 10. In a preferred embodiment the Tsp gene ismutated to insert an Ase I restriction site to create a third in-framestop codon at codon 21, as shown in FIG. 10.

In a preferred embodiment the knockout mutated Tsp gene has the DNAsequence of SEQ ID NO: 25, which includes the 6 nucleotides ATGAATupstream of the start codon. The mutations which have been made in theknockout mutated Tsp sequence of SEQ ID NO: 25 are shown in FIG. 10. Inone embodiment the mutated Tsp gene has the DNA sequence of nucleotides7 to 2048 of SEQ ID NO: 25.

In embodiments of the present invention the cell comprises a mutatedDegP gene. As used herein, “DegP” means a gene encoding DegP protein(also known as HtrA), which has dual function as a chaperone and aprotease. The sequence of the wild-type DegP gene is shown in SEQ ID NO:29 and the sequence of the non-mutated DegP protein is shown in SEQ IDNO: 30.

At low temperatures DegP functions as a chaperone and at hightemperatures DegP has a preference to function as a protease.

In the embodiments where the cell comprises the DegP mutation, the DegPmutation in the cell provides a mutated DegP gene encoding a DegPprotein having chaperone activity but not full protease activity.

The expression “having chaperone activity” in the context of the presentinvention means that the mutated DegP protein has the same orsubstantially the same chaperone activity compared to the wild-typenon-mutated DegP protein. Preferably, the mutated DegP gene encodes aDegP protein having 50% or more, 60% or more, 70% or more, 80% or more,90% or more or 95% or more of the chaperone activity of a wild-typenon-mutated DegP protein. More preferably, the mutated DegP gene encodesa DegP protein having the same chaperone activity compared to wild-typeDegP.

The expression “having reduced protease activity” in the context of thepresent invention means that the mutated DegP protein does not have thefull protease activity compared to the wild-type non-mutated DegPprotein. Preferably, the mutated DegP gene encodes a DegP protein having50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% orless of the protease activity of a wild-type non-mutated DegP protein.More preferably, the mutated DegP gene encodes a DegP protein having noprotease activity. The cell is not deficient in chromosomal DegP, i.e.,the DegP gene sequence has not been deleted or mutated to preventexpression of any form of DegP protein.

Any suitable mutation may be introduced into the DegP gene in order toproduce a protein having chaperone activity and reduced proteaseactivity. The protease and chaperone activity of a DegP proteinexpressed from a gram-negative bacterium may be easily tested by aperson skilled in the art by any suitable method, e.g., wherein theprotease and chaperone activities of DegP are tested on MalS, a naturalsubstrate of DegP.

DegP is a serine protease and has an active center consisting of acatalytic triad of amino acid residues of His105, Asp135 and Ser210. TheDegP mutation to produce a protein having chaperone activity and reducedprotease activity may comprise a mutation, such as a missense mutation,affecting one, two or three of His105, Asp135 and Ser210.

Accordingly, the mutated DegP gene may comprise:

-   -   a mutation affecting the amino acid His105;    -   a mutation affecting the amino acid Asp135;    -   a mutation affecting the amino acid Ser210;    -   a mutation affecting the amino acids His105 and Asp135;    -   a mutation affecting the amino acids His105 and Ser210;    -   a mutation affecting the amino acids Asp135 and Ser210; or    -   a mutation affecting the amino acids His105, Asp135 and Ser210.

One, two or three of His105, Asp135 and Ser210 may be changed to anysuitable amino acid which results in a protein having chaperone activityand reduced protease activity. For example, one, two or three of His105,Asp135 and Ser210 may be changed to a small amino acid such as Gly orAla. A further suitable mutation is to change one, two or three ofHis105, Asp135 and Ser210 to an amino acid having opposite properties,such as Asp135 being changed to Lys or Arg, polar His105 being changedto a non-polar amino acid such as Gly, Ala, Val or Leu and smallhydrophilic Ser210 being changed to a large or hydrophobic residue suchas Val, Leu, Phe or Tyr. Preferably, the DegP gene comprises thealteration S210A, as shown in FIG. 11, which has been found to produce aprotein having chaperone activity but not protease activity.

DegP has two PDZ domains, PDZ1 (residues 260-358) and PDZ2 (residues359-448), which mediate protein-protein interaction. In one embodimentof the present invention the DegP gene is mutated to delete the PDZ1domain and/or the PDZ2 domain. The deletion of PDZ1 and PDZ2 results incomplete loss of protease activity of the DegP protein and loweredchaperone activity compared to wild-type DegP protein, while deletion ofeither PDZ1 or PDZ2 results in 5% protease activity and similarchaperone activity compared to wild-type DegP protein.

The mutated DegP gene may also comprise a silent non-naturally occurringrestriction site, such as Ase I, in order to aid in identification andscreening methods, for example as shown in FIG. 11.

The preferred sequence of the mutated DegP gene comprising the pointmutation S210A and an Ase I restriction marker site is provided in SEQID NO: 31 and the encoded protein sequence is shown in SEQ ID NO: 27.The mutations which have been made in the mutated DegP sequence of SEQID NO: 32 are shown in FIG. 11.

In the embodiments of the present invention wherein the cell comprises amutated DegP gene encoding a DegP protein having chaperone activity andreduced protease activity, one or more of the cells provided by thepresent invention may provide an improved yield of correctly foldedproteins from the cell relative to mutated cells wherein the DegP genehas been mutated to knockout DegP, preventing DegP expression, such aschromosomal deficient DegP. In a cell comprising a knockout mutated DegPgene preventing DegP expression, the chaperone activity of DegP is lostcompletely, whereas in the cell according to the present invention thechaperone activity of DegP is retained while the full protease activityis lost. In these embodiments, one or more cells according to thepresent invention have a lower protease activity to prevent proteolysisof the protein while maintaining the chaperone activity to allow correctfolding and transportation of the protein in the host cell.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated OmpT gene, such asbeing deficient in chromosomal OmpT (SEQ ID NO: 39).

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated DegP gene, such asbeing deficient in chromosomal DegP. In one embodiment the gram-negativebacterial cell according to the present invention does not carry amutated DegP gene.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a knockout mutated ptr gene, such asbeing deficient in chromosomal ptr.

In one embodiment the gram-negative bacterial cell according to thepresent invention does not carry a mutated spr gene.

Any suitable gram-negative bacterium may be used as the parental cellfor producing the recombinant cell of the present invention. Suitablegram-negative bacterium include Salmonella typhimurium, Pseudomonasfluorescens, Envinia carotovora, Shigella, Klebsiella pneumoniae,Legionella pneumophila, Pseudomonas aeruginosa, Acinetobacter baumanniiand E. coli. Preferably the parental cell is E. coli. Any suitablestrain of E. coli may be used in the present invention but preferably awild-type W3110 strain, such as K-12 W3110, is used.

A drawback associated with strains previously created and used toexpress recombinant proteins involves the use of mutations of genesinvolved in cell metabolism and DNA replication, such as phoA, fhuA,lac, rec, gal, ara, arg, thi and pro in E. coli strains. These mutationsmay have many deleterious effects on the host cell including effects oncell growth, stability, periplasmic leakage, recombinant proteinexpression yield and toxicity. Strains having one or more of thesegenomic mutations, particularly strains having a high number of thesemutations, may exhibit a loss of fitness which reduces bacterial growthrate to a level which is not suitable for industrial protein production.Further, any of the above genomic mutations may affect other genes incis and/or in trans in unpredictable harmful ways, thereby altering thestrain's phenotype, fitness and protein profile. Further, the use ofheavily mutated cells is not generally suitable for producingrecombinant proteins for commercial use, particularly therapeutics,because such strains generally have defective metabolic pathways andhence may grow poorly or not at all in minimal or chemically definedmedia.

In a preferred embodiment, the cells carry only the minimal mutations tointroduce the recombinant polynucleotide encoding DsbC and the one ormore polynucleotides encoding the antibody or antigen-binding fragmentthereof and optionally a mutation resulting in reduced Tsp proteaseactivity and optionally an spr gene or a mutant thereof.

In one embodiment wherein the polynucleotide encoding DsbC and/or theone or more polynucleotides encoding the antibody or antigen-bindingfragment thereof are inserted into the cell's genome only minimalmutations are made to the cell's genome to introduce the recombinantpolynucleotide encoding DsbC and/or the antibody. In a furtherembodiment wherein the recombinant polynucleotide encoding DsbC and thepolynucleotide encoding the antibody are present in the same ordifferent expression vectors, the genome is preferably isogenic to awild-type cell genome.

In one embodiment the cells do not carry any other mutations which mayhave deleterious effects on the cell's growth and/or ability to expressa protein of interest. Accordingly, one or more of the recombinant hostcells of the present invention may exhibit improved protein expressionand/or improved growth characteristics compared to cells comprisinggenetically engineered mutations to the genomic sequence. The cellsprovided by the present invention are also more suitable for use toproduce therapeutic proteins compared to cells comprising disruptions tothe cell genome.

In a preferred embodiment, the cell is isogenic to a wild-type E. colicell except for the recombinant polynucleotide encoding DsbC and the oneor more polynucleotides encoding an antibody or antigen-binding fragmentthereof specifically binding to CD154 and optionally a mutationresulting in reduced Tsp protease activity and optionally an spr gene ora mutant thereof.

In one embodiment there is provided a cell isogenic to an E. coli strainW3110 except with reduced Tsp activity and an spr gene or a mutantthereof, for use with a plasmid suitable for expressing DsbC and anantibody or an antigen-binding fragment thereof specifically binding toCD154.

More preferably the cell according to the present invention is isogenicto an E. coli strain W3110 except for the recombinant polynucleotideencoding DsbC and the one or more polynucleotides encoding an antibodyor an antigen-binding fragment thereof specifically binding to CD154.

The cell provided by the present invention comprises one or morepolynucleotides encoding an antigen-binding antibody fragment withspecificity for CD154.

A cell comprising as employed herein is intended to refer to where theentity concerned is integrated into the cell's genome or where the cellcontains a vector such as a plasmid containing and generally forexpressing the entity.

The antibody or antibody fragment may be multi-valent, multi-specific,humanized, fully human or chimeric. The antibody or antibody fragmentcan be from any species but is preferably derived from a monoclonalantibody, a human antibody, or a humanized fragment. The antibodyfragment can be derived from any class (e.g., IgG, IgE, IgM, IgD or IgA)or subclass of immunoglobulin molecule and may be obtained from anyspecies including, for example, mouse, rat, shark, rabbit, pig, hamster,camel, llama, goat or human. Parts of the antibody fragment may beobtained from more than one species; for example the antibody fragmentsmay be chimeric. In one example the constant regions are from onespecies and the variable regions from another.

The antibody fragment may be a VH, VL, VHH, Fab, modified Fab, Fab′,F(ab′)₂ or Fv fragment; a light chain or heavy chain monomer or dimer;or a diabody, triabody, tetrabody, minibody, domain antibody orsingle-chain antibody, e.g., a single-chain Fv in which the heavy andlight chain variable domains are joined by a peptide linker, Fab-Fv, ordual specificity antibody, such as a Fab-dAb, as described in WO2009/040562. Similarly, the heavy and light chain variable regions maybe combined with other antibody domains as appropriate. Antibodyfragments are known in the art (Holliger and Hudson 1126-36).

The antibody specifically binding to CD154 is preferably the antibody342 described in WO 2008/118356 (the contents of which are incorporatedherein by reference) or comprises the CDRs or variable heavy and lightchain regions of the antibody 342 described in WO 2008/118356. Theantibody fragment specifically binding to CD154 is preferably derivedfrom the antibody 342 described in WO 2008/118356 and/or comprises theCDRs or variable heavy and light chain regions of said antibody.

In a one embodiment the antibody or antibody fragment specificallybinding to CD154 comprises a heavy chain wherein the variable domaincomprises three CDRs, wherein the CDRs are selected from SEQ ID NO: 1for CDRH1, SEQ ID NO: 2 for CDRH2 and SEQ ID NO: 3 for CDRH3.

In one embodiment the antibody or antibody fragment specifically bindingto CD154 comprises a light chain wherein the variable domain comprisesthree CDRs, wherein the CDRs are selected from SEQ ID NO: 4 for CDRL1,SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3.

In one embodiment the antibody or antibody fragment specifically bindingto CD154 comprises a heavy chain comprising the sequence of SEQ ID NO: 1for CDRH1, the sequence of SEQ ID NO: 2 for CDRH2 and the sequence ofSEQ ID NO: 3 for CDRH3.

In one embodiment the antibody or antibody fragment specifically bindingto CD154 comprises a light chain comprising the sequence of SEQ ID NO: 4for CDRL1, the sequence of SEQ ID NO: 5 for CDRL2 and the sequence ofSEQ ID NO: 6 for CDRL3.

In one embodiment the antibody or antibody fragment specifically bindingto CD154 comprises a heavy chain comprising the sequence of SEQ ID NO: 1for CDRH1, the sequence of SEQ ID NO: 2 for CDRH2 and the sequence ofSEQ ID NO: 3 for CDRH3 and a light chain comprising the sequence of SEQID NO: 4 for CDRL1, the sequence of SEQ ID NO: 5 for CDRL2 and thesequence of SEQ ID NO: 6 for CDRL3.

The antibody is preferably a CDR-grafted antibody molecule and typicallythe variable domain comprises human acceptor framework regions andnon-human donor CDRs.

Preferably, the antibody comprises the light chain variable domain (SEQID NO: 7) and the heavy chain variable domain (SEQ ID NO: 9).

Preferably the antibody is a Fab fragment. Preferably the Fab fragmenthas a heavy chain comprising or consisting of the sequence given as SEQID NO: 14 and a light chain comprising or consisting of the sequencegiven as SEQ ID NO: 12. The amino acid sequences given in SEQ ID NO: 14and SEQ ID NO: 12 are preferably encoded by the nucleotide sequencesgiven in SEQ ID NO: 13 and SEQ ID NO: 11, respectively.

Alternatively, it is preferred that the antibody fragment is a modifiedFab fragment wherein the modification is the addition to the C-terminalend of its heavy chain one of or more amino acids to allow theattachment of an effector or reporter molecule. Preferably, theadditional amino acids form a modified hinge region containing one ortwo cysteine residues to which the effector or reporter molecule may beattached as known in the art (see, e.g., WO 98/25971, which isincorporated herein in its entirety).

The cell according to the present invention comprises a DNA sequenceencoding the antibody. Preferably, the DNA sequence encodes the heavyand the light chain of the antibody.

In one preferred embodiment, the DNA sequence encodes a light chain andcomprises the sequence shown herein.

The DNA sequence of the present invention may comprise synthetic DNA,for instance produced by chemical processing, cDNA, genomic DNA or anycombination thereof.

The constant region domains of the antibody, if present, may be selectedwith regard to the proposed function of the antibody molecule, and inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgD, IgE, IgG or IgMdomains. In particular, human IgG constant region domains may be used,especially of the IgG₁ and IgG₃ isotypes when the antibody molecule isintended for therapeutic uses and antibody effector functions arerequired. Alternatively, IgG₂ and IgG₄ isotypes may be used when theantibody molecule is intended for therapeutic purposes and antibodyeffector functions are not required, e.g., for simply blocking CD154activity.

The antibody may be useful in the treatment of diseases or disordersincluding inflammatory diseases and disorders, immune disease anddisorders, fibrotic disorders and cancers.

The terms “inflammatory disease” or “autoimmune disorder” and “immunedisease or disorder” include rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, juvenile arthritis, Still's disease, Hashimoto'sthyroiditis, Graves' disease, Sjögren's syndrome, Goodpasture'ssyndrome, Addison's disease, vasculitis including ANCA-associatedvasculitis and Wegener's granulomatosis, primary biliary cirrhosis,sclerosing cholangitis, autoimmune hepatitis polymyalgia rheumatica,Guillain-Barre syndrome, antiphospholipid syndrome, idiopathicthrombocytopaenia, autoimmune haemolytic anaemia, pernicious anaemia,pemphigus vulgaris, dermatomyositis, bullous pemphigoid,Henoch-Schönlein purpura, Muckle-Wells syndrome, psoriasis, Crohn'sdisease, ulcerative colitis, SLE (Systemic Lupus Erythematosus), celiacdisease, asthma, allergic rhinitis, atopic dermatitis, multiplesclerosis, Type I diabetes mellitus, transplantation andgraft-versus-host disease.

The term “fibrotic disorder” as used herein refers to a disordercharacterized by the formation or development of excess fibrousconnective tissue in an organ or tissue, frequently as a reparative orreactive process. A fibrotic disorder can affect single organs, such asthe lungs (for example, without limitation, idiopathic pulmonaryfibrosis, interstitial lung disease), the liver, the intestine, thekidney, the heart or the skin, or affect multiple organs, for example,without limitation, systemic sclerosis. The term “fibrotic disorder”also relates to scarring of the skin. Scars of the skin include, but arenot limited to, keloid scars, contracture scars that occur, for example,without limitation, after skin burn, hypertrophic scars and acne scars.

The host cell of the invention may also comprise further polynucleotidesencoding one or more further proteins of interest.

The recombinant gram-negative bacterial cell according to the presentinvention may be produced by any suitable means.

The skilled person knows suitable techniques which may be used to insertthe recombinant polynucleotide encoding DsbC and the polynucleotideencoding the antibody. The recombinant polynucleotide encoding DsbCand/or the polynucleotide encoding the antibody may be integrated intothe cell's genome using a suitable vector such as pKO3, described inLink et al., which is incorporated herein by reference in its entirety(Link, Phillips, and Church 6228-37).

Alternatively or additionally, the recombinant polynucleotide encodingDsbC and/or the polynucleotide encoding the antibody may benon-integrated in a recombinant expression cassette. In one embodimentan expression cassette is employed in the present invention to carry thepolynucleotide encoding DsbC and/or the polynucleotide encoding theantibody, which typically comprises a protein coding sequence encodingDsbC, one or more protein coding sequences encoding the antibody and oneor more regulatory expression sequences. The one or more regulatoryexpression sequences may include a promoter. The one or more regulatoryexpression sequences may also include a 3′ untranslated region such as atermination sequence. Suitable promoters are discussed in more detailbelow.

In one embodiment the gene encoding DsbC and/or the antibody or fragmentthereof is/are integrated into the genome of the host cell to create astable cell line.

In one embodiment, the cell according to the present invention comprisesone or more expression vectors, such as plasmids. The vector preferablycomprises one or more of the expression cassettes as defined above. Thehost cell preferably comprises an expression vector comprising DNAencoding an antibody or an antigen-binding fragment thereof specificallybinding to CD154 as described above. Preferably the expression vectorcomprises a polynucleotide sequence encoding a light chain and apolynucleotide sequence encoding a heavy chain of the antibody or anantigen-binding fragment thereof specifically binding to CD154.

In a preferred embodiment, the expression vector is an E. coliexpression vector.

In one embodiment the polynucleotide sequence encoding the antibody andthe polynucleotide sequence encoding DsbC are inserted into separateexpression vectors.

For production of products comprising both heavy and light chains, thecell line may be transformed with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, the polynucleotide sequence encoding the antibody and thepolynucleotide encoding DsbC are inserted into one vector. Preferablythe vector comprises the sequences encoding the light and heavy chainpolypeptides of the antibody.

The present invention also provides an expression vector comprising arecombinant polynucleotide encoding DsbC and an antibody or anantigen-binding fragment thereof specifically binding to CD154. Theexpression vector is a multi-cistronic vector comprising thepolynucleotide sequence encoding DsbC and the polynucleotide sequenceencoding the antibody.

The multicistronic vector may be produced by an advantageous cloningmethod which allows repeated sequential cloning of polynucleotidesequences into a vector. The method uses compatible cohesive ends of apair of restriction sites, such as the “AT” ends of AseI and NdeIrestriction sites. A polynucleotide comprising a coding sequence andhaving compatible cohesive ends, such as an AseI-NdeI fragment, may becloned into a restriction site in the vector, such as NdeI. Theinsertion of the polynucleotide sequence destroys the 5′ restrictionsite but creates a new 3′ restriction site, such as NdeI, which may thenbe used to insert a further polynucleotide sequence comprisingcompatible cohesive ends. The process may then be repeated to insertfurther sequences. Each polynucleotide sequence inserted into the vectorcomprises a non-coding sequence 3′ to the stop codon which may comprisean Ssp I site for screening, a Shine-Dalgarno ribosome binding sequence,an A-rich spacer and an NdeI site encoding a start codon.

A diagrammatic representation of the creation of a vector comprising apolynucleotide sequence encoding a light chain of an antibody (LC), aheavy chain of an antibody (HC), a DsbC polynucleotide sequence and afurther polynucleotide sequence is shown in FIG. 1.

The cell according to the present invention preferably comprises anexpression vector as defined above.

In the embodiment wherein the cell also expresses one or more furtherproteins as follows:

-   -   one or more proteins capable of facilitating protein folding,        such as FkpA, Skp, SurA, PPiA and PPiD;    -   one or more proteins capable of facilitating protein secretion        or translocation, such as SecY, SecE, SecG, SecYEG, SecA, SecB,        FtsY and Lep; and/or    -   one or more proteins capable of facilitating disulfide bond        formation, such as DsbA, DsbB, DsbD, DsbG,

the one or more further proteins may be expressed from one or morepolynucleotides inserted into the same vector as the polynucleotideencoding DsbC and/or the one or more polynucleotides encoding theantibody or antigen-binding fragment thereof specifically binding toCD154. Alternatively, the one or more polynucleotides may be insertedinto separate vectors.

The expression vector may be produced by inserting one or moreexpression cassettes as defined above into a suitable vector.Alternatively, the regulatory expression sequences for directingexpression of the polynucleotide sequence may be contained in theexpression vector and thus only the encoding region of thepolynucleotide may be required to complete the expression vector.

The polynucleotide encoding DsbC and/or the polynucleotide encoding theantibody or antigen-binding fragment thereof specifically binding toCD154 is suitably inserted into a replicable vector, typically anautonomously replicating expression vector, for expression in the cellunder the control of a suitable promoter for the cell. Many vectors areknown in the art for this purpose and the selection of the appropriatevector may depend on the size of the nucleic acid and the particularcell type.

Examples of expression vectors which may be employed to transform thehost cell with a polynucleotide according to the invention include:

-   -   a plasmid, such as pBR322 or pACYC184;    -   a viral vector such as a bacterial phage; and/or    -   a transposable genetic element such as a transposon.

Such expression vectors usually comprise a plasmid origin of DNAreplication, an antibiotic selectable marker, a promoter andtranscriptional terminator separated by a multi-cloning site (expressioncassette) and a DNA sequence encoding a ribosome binding site.

The promoters employed in the present invention can be linked to therelevant polynucleotide directly or can alternatively be located in anappropriate position, for example in a vector such that when therelevant polypeptide is inserted the relevant promoter can act on thesame. In one embodiment the promoter is located before the encodingportion of the polynucleotide on which it acts, for example a relevantpromoter before each encoding portion of polynucleotide. “Before” asused herein is intended to imply that the promoter is located at the 5′end in relation to the encoding polynucleotide portion.

The promoters may be endogenous or exogenous to the host cells. Suitablepromoters include lac, tac, trp, phoA, Ipp, Arab, tet and T7.

One or more promoters employed may be inducible promoters. In theembodiment wherein the polynucleotide encoding DsbC and thepolynucleotide encoding the antibody are inserted into one vector, thenucleotide sequences encoding DsbC and the antibody may be under thecontrol of a single promoter or separate promoters. In the embodimentwherein the nucleotide sequences encoding DsbC and the antibody areunder the control of separate promoters, the promoters may beindependently inducible promoters.

Promoters for use in bacterial systems also generally contain aShine-Dalgamo (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA.

The expression vector preferably also comprises a dicistronic messagefor producing the antibody or antigen-binding fragment thereof asdescribed in WO 03/048208 or WO 2007/039714 (the contents of which areincorporated herein by reference in their entirety). Preferably theupstream cistron contains DNA coding for the light chain of the antibodyand the downstream cistron contains DNA coding for the correspondingheavy chain, and the dicistronic intergenic sequence (IGS) preferablycomprises a sequence selected from IGS1 (SEQ ID NO: 33), IGS2 (SEQ IDNO: 34), IGS3 (SEQ ID NO: 35) and IGS4 (SEQ ID NO: 36).

A preferable expression vector comprises a tricistronic message forproducing the light chain and the heavy chain of the antibody orantigen-binding fragment thereof as described above and a message forproducing the recombinant DsbC, preferably comprising a his-tag.

The terminators may be endogenous or exogenous to the host cells. Asuitable terminator is rrnB.

Further suitable transcriptional regulators including promoters andterminators and protein targeting methods may be found in Makrides etal., which is incorporated herein by reference in its entirety (Makrides512-38).

The DsbC polynucleotide inserted into the expression vector preferablycomprises the nucleic acid encoding the DsbC signal sequence and theDsbC coding sequence. The DsbC protein may also be directed to theperiplasm by genetic fusion to other signal peptides, for example thosefrom the proteins OmpA, MalB, PelB, PhoA, PhoS, LppA, and DsbA. Thevector preferably contains a nucleic acid sequence that enables thevector to replicate in one or more selected host cells, preferably toreplicate independently of the host chromosome. Such sequences arewell-known for a variety of bacteria.

In one embodiment the DsbC and/or the protein of interest comprises ahistidine-tag at the N-terminus and/or C-terminus.

The antibody molecule may be secreted from the cell or targeted to theperiplasm by suitable signal sequences. Alternatively, the antibodymolecules may accumulate within the cell's cytoplasm. Preferably theantibody molecule is targeted to the periplasm.

The polynucleotide encoding the antibody may be expressed as a fusionwith another polypeptide, preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature polypeptide. The heterologous signal sequence selected should beone that is recognized and processed by the host cell. For prokaryotichost cells that do not recognize and process the native or a eukaryoticpolypeptide signal sequence, the signal sequence is substituted by aprokaryotic signal sequence. Suitable signal sequences include OmpA,PhoA, LamB, PelB, DsbA and DsbC. In an embodiment where the cellcomprises a polynucleotide encoding a heavy chain of the antibody and apolynucleotide encoding a light chain of the antibody, eachpolynucleotide may comprise a signal sequence, such as OmpA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required. General methods by whichthe vectors may be constructed, transfection methods and culture methodsare well-known to those skilled in the art.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibody. Desired DNA sequences may besynthesised completely or in part using oligonucleotide synthesistechniques. Site-directed mutagenesis and polymerase chain reaction(PCR) techniques may be used as appropriate.

Embodiments of the invention described herein with reference to thepolynucleotide apply equally to alternative embodiments of theinvention, for example vectors, expression cassettes and/or host cellscomprising the components employed therein, as far as the relevantaspect can be applied to same.

The present invention also provides a method for producing an antibodyor an antigen-binding fragment thereof specifically binding to CD154comprising:

culturing a recombinant gram-negative bacterial cell as defined above ina culture medium under conditions effective to express the antibody orthe antigen-binding fragment thereof specifically binding to CD154 andthe recombinant polynucleotide encoding DsbC; and

recovering the antibody or an antigen-binding fragment thereofspecifically binding to CD154 from the periplasm of the recombinantgram-negative bacterial cell and/or the culture medium.

The gram-negative bacterial cell and antibody preferably employed in themethod of the present invention are described in detail above.

The recombinant polynucleotide encoding DsbC and the polynucleotideencoding the antibody or antigen-binding fragment thereof specificallybinding to CD154 may be incorporated into the host cell using anysuitable means known in the art. As discussed above, typically thepolynucleotide encoding DsbC and the polynucleotide encoding theantibody are incorporated as part of the same or separate expressionvectors which are transformed into the cell.

The polynucleotide encoding DsbC and the polynucleotide encoding theantibody or antigen-binding fragment thereof specifically binding toCD154 can be transformed into a cell using standard techniques, forexample employing rubidium chloride, PEG or electroporation.

The method according to the present invention may also employ aselection system to facilitate selection of stable cells which have beensuccessfully transformed with the polynucleotide encoding the protein ofinterest. The selection system typically employs co-transformation of apolynucleotide encoding a selection marker. In one embodiment, eachpolynucleotide transformed into the cell further comprises apolynucleotide encoding one or more selection markers. Accordingly, thetransformation of the polynucleotides encoding DsbC and the antibody orantigen-binding fragment thereof specifically binding to CD154 and theone or more polynucleotides encoding the marker occurs together and theselection system can be employed to select those cells which produce thedesired proteins.

Cells able to express the one or more markers are able tosurvive/grow/multiply under certain artificially imposed conditions, forexample the addition of a toxin or antibiotic, because of the propertiesendowed by the polypeptide/gene or polypeptide component of theselection system incorporated therein (e.g., antibiotic resistance).Those cells that cannot express the one or more markers are not able tosurvive/grow/multiply in the artificially imposed conditions. Theartificially imposed conditions can be chosen to be more or lessvigorous, as required.

Any suitable selection system may be employed in the present invention.Typically the selection system may be based on including in the vectorone or more genes that provide resistance to a known antibiotic, forexample a tetracycline, chloramphenicol, kanamycin or ampicillinresistance gene. Cells that grow in the presence of a relevantantibiotic can be selected as they express both the gene that givesresistance to the antibiotic and the desired protein.

An inducible expression system or a constitutive promoter may be used inthe present invention to express the antibody and/or the DsbC. Suitableinducible expression systems and constitutive promoters are well-knownin the art.

In one embodiment wherein the polynucleotide encoding DsbC and thepolynucleotide encoding the antibody are under the control of the sameor separate inducible promoters, the expression of the polynucleotide(s)encoding the antibody and the recombinant polynucleotide encoding DsbCis induced by adding an inducer to the culture medium.

Any suitable medium may be used to culture the transformed cell. Themedium may be adapted for a specific selection system, for example themedium may comprise an antibiotic, to allow only those cells which havebeen successfully transformed to grow in the medium.

The cells obtained from the medium may be subjected to further screeningand/or purification as required. The method may further comprise one ormore steps to extract and purify the protein of interest as required.

The antibody or antigen-binding fragment thereof may be recovered andpurified from the strain, including from the cytoplasm, periplasm, orsupernatant. Suitable methods include fractionation on immunoaffnity orion-exchange columns; ethanol precipitation; reversed-phase HPLC;hydrophobic-interaction chromatography; chromatography on silica;chromatography on an ion-exchange resin such as S-SEPHAROSE and DEAE;chromatofocusing; ammonium-sulfate precipitation; and gel filtration.

In one embodiment the method further comprises separating the antibodyor antigen-binding fragment thereof from DsbC.

Antibodies or antigen-binding fragments thereof may be suitablyseparated from the culture medium, cytoplasm extract and/or periplasmextract by conventional antibody purification procedures such as proteinA-Sepharose, protein G chromatography, protein L chromatography,thiophilic, mixed mode resins, His-tag, FLAGTag, hydroxylapatitechromatography, gel electrophoresis, dialysis, affinity chromatography,ammonium sulfate, ethanol or PEG fractionation/precipitation, ionexchange membranes, expanded bed adsorption chromatography (EBA) orsimulated moving bed chromatography.

The method may also include a further step of measuring the quantity ofexpression of the protein of interest and selecting cells having highexpression levels of the protein of interest.

One or more method steps described herein may be performed incombination in a suitable container such as a bioreactor.

After expression, the antibody may be further processed, for example byconjugation to another entity such as an effector molecule. Accordingly,the method according to the present invention may comprise a furtherstep of attaching an effector molecule to the antibody.

The term “effector molecule” as used herein includes, for example,antineoplastic agents, drugs, toxins (such as enzymatically activetoxins of bacterial or plant origin and fragments thereof, e.g., ricinand fragments thereof), biologically active proteins, for exampleenzymes, other antibodies or antibody fragments, synthetic or naturallyoccurring polymers, nucleic acids and fragments thereof, e.g., DNA, RNAand fragments thereof, radionuclides, particularly radio-iodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy. Effector molecules may be attached to the antibody orfragment thereof by any suitable method; for example an antibodyfragment may be modified to attach at least one effector molecule asdescribed in WO 2005/003171 or WO 2005/003170 (the contents of which areincorporated herein by reference in their entirety). WO 2005/003171 andWO 2005/003170 also describe suitable effector molecules.

The antibody may have a macrocycle, for chelating a heavy metal atom, ora toxin, such as ricin, attached to it by a covalent bridging structure.Alternatively, procedures of recombinant DNA technology may be used toproduce an antibody molecule in which the Fc fragment (CH2, CH3 andhinge domains), the CH2 and CH3 domains or the CH3 domain of a completeimmunoglobulin molecule has/have been replaced by, or has/have attachedthereto by peptide linkage, a functional non-immunoglobulin protein,such as an enzyme or toxin molecule. In the embodiment wherein theantibody is a modified Fab fragment having at the C-terminal end of itsheavy chain one or more amino acids to allow attachment of an effectoror reporter molecule, the additional amino acids preferably form amodified hinge region containing one or two cysteine residues to whichthe effector or reporter molecule may be attached.

A preferred effector group is a polymer molecule, which may be attachedto the modified Fab fragment to increase its half-life in vivo.

The polymer molecule may, in general, be a synthetic or a naturallyoccurring polymer, for example an optionally substituted straight orbranched chain polyalkylene, polyalkenylene or polyoxyalkylene polymeror a branched or unbranched polysaccharide, e.g., a homo- orhetero-polysaccharide.

Particular optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol),poly(propyleneglycol) poly(vinyl alcohol) or derivatives thereof,especially optionally substituted poly(ethyleneglycol) such asmethoxypoly(ethyleneglycol) or derivatives thereof. Particular naturallyoccurring polymers include lactose, amylose, dextran, glycogen orderivatives thereof. “Derivatives” as used herein is intended to includereactive derivatives, for example thiol-selective reactive groups suchas maleimides and the like. The reactive group may be linked directly orthrough a linker segment to the polymer. It will be appreciated that theresidue of such a group will in some instances form part of the productas the linking group between the antibody fragment and the polymer.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50,000 Da,preferably from 5000 Da to 40,000 Da and more preferably from 25,000 Dato 40,000 Da. The polymer size may in particular be selected on thebasis of the intended use of the product. Thus, for example, where theproduct is intended to leave the circulation and penetrate tissue, forexample for use in the treatment of an inflammation, it may beadvantageous to use a small molecular weight polymer, for example with amolecular weight of around 5000 Da. For applications where the productremains in the circulation, it may be advantageous to use a highermolecular weight polymer, for example having a molecular weight in therange from 25,000 Da to 40,000 Da.

Particularly preferred polymers include a polyalkylene polymer, such asa poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) ora derivative thereof, and especially with a molecular weight in therange from about 25,000 Da to about 40,000 Da.

Each polymer molecule attached to the modified antibody fragment may becovalently linked to the sulfur atom of a cysteine residue located inthe fragment. The covalent linkage will generally be a disulfide bondor, in particular, a sulfur-carbon bond.

Where desired, the antibody fragment may have one or more effector orreporter molecules attached to it. The effector or reporter moleculesmay be attached to the antibody fragment through any available aminoacid side-chain or terminal amino acid functional group located in thefragment, for example any free amino, imino, hydroxyl or carboxyl group.One or more effector or reporter molecules may be attached to an aminoacid at or towards the C-terminal end of the heavy chain and/or thelight chain of the antibody.

An activated polymer may be used as the starting material in thepreparation of polymer-modified antibody fragments as described above.The activated polymer may be any polymer containing a thiol reactivegroup such as an α-halocarboxylic acid or ester, e.g., iodoacetamide, animide, e.g., maleimide, a vinyl sulfone or a disulfide. Such startingmaterials may be obtained commercially (for example from ShearwaterPolymers, Inc., Huntsville, Ala., USA) or may be prepared fromcommercially available starting materials using conventional chemicalprocedures.

Where it is desired to obtain an antibody fragment linked to an effectoror reporter molecule, this may be prepared by standard chemical orrecombinant DNA procedures in which the antibody fragment is linkedeither directly or via a coupling agent to the effector or reportermolecule either before or after reaction with the activated polymer asappropriate. Particular chemical procedures include, for example, thosedescribed in WO 93/62331 and WO 92/22583. Alternatively, where theeffector or reporter molecule is a protein or polypeptide the linkagemay be achieved using recombinant DNA procedures, for example asdescribed in WO 86/01533 and EP-A-0392745.

Preferably, the modified Fab fragment provided by the method of thepresent invention is PEGylated (i.e., has PEG (poly(ethyleneglycol))covalently attached thereto) according to the method disclosed inEP-A-0948544. Preferably the antibody is a PEGylated modified Fabfragment as shown in FIG. 2. As shown in FIG. 2, the modified Fabfragment has attached to one of the cysteine residues at the C-terminalend of the modified hinge region of the heavy chain alysyl-maleimide-derived group wherein each of the two amino groups ofthe lysyl residue has covalently linked to it amethoxypoly(ethyleneglycol) residue having a molecular weight of about20,000 Da, such that the total average molecular weight of themethoxypoly(ethyleneglycol) residues is about 40,000 Da; more preferablythe lysyl-maleimide-derived group is[1-[[[2-[[3-(2,5-dioxo-1-pyrrolidinyl)-1-oxopropyl]amino]ethyl]amino]-carbonyl]-1,5-pentanediyl]bis(iminocarbonyl).A lysine residue is covalently linked to the maleimide group. To each ofthe amine groups on the lysine residue is attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the entireeffector molecule is therefore approximately 40,000 Da.

Accordingly, the method according to the present invention preferablycomprises attaching to one of the cysteine residues at the C-terminalend of the heavy chain a lysyl-maleimide group wherein each amino groupof the lysyl residue has covalently linked to it amethoxypoly(ethyleneglycol) residue having a molecular weight of about20,000 Da.

In one embodiment a physical property of a contaminating host protein isaltered by the addition of an amino acid tag to the C-terminus orN-terminus. In a preferred embodiment the physical property that isaltered is the isoelectric point and the amino acid tag is apoly-aspartic acid tag attached to the C-terminus. In one embodiment theE. coli proteins altered by the addition of said tag are dipeptidebinding protein (DppA), maltose binding protein (MBP), thioredoxin andphosphate binding protein (PhoS/PstS). In one specific embodiment the pIof the E. coli phosphate binding protein (PhoS/PstS) is reduced from 7.2to 5.1 by the addition of a poly-aspartic acid tag (polyD) containing 6aspartic acid residues to the C-terminus.

Also preferred is the modification of specific residues of thecontaminating E. coli protein to alter its physical properties, eitheralone or in combination with the addition of N- or C-terminal tags. Suchchanges can include insertions or deletions to alter the size of theprotein or amino acid substitutions to alter pI or hydrophobicity. Inone embodiment these residues are located on the surface of the protein.In a preferred embodiment surface residues of the PhoS protein arealtered in order to reduce the pI of the protein. Preferably residuesthat have been implicated to be important in phosphate binding (U.S.Pat. No. 5,304,472) are avoided in order to maintain a functional PhoSprotein.

Examples

Cell Lines

For all experiments the E. coli cell line W3110 was used as the parentalwild-type cell line.

Cell lines were created carrying the following mutations:

a) a mutated Tsp gene;

b) a mutated Tsp gene and carrying recombinant DsbC;

c) a mutated Tsp gene and a mutated spr gene; and

d) a mutated Tsp gene, a mutated spr gene and carrying recombinant DsbC.

Example 1 Generation of Cell Line Carrying Mutated Tsp Gene MXE001(ΔTsp)

The MXE001 Strain was Generated as Follows:

The Tsp cassette was moved as Sal I, Not I restriction fragments intosimilarly restricted pKO3 plasmids. The pKO3 plasmid uses thetemperature-sensitive mutant of the pSC101 origin of replication (RepA)along with a chloramphenicol marker to force and select for chromosomalintegration events. The sacB gene which encodes for levansucrase islethal to E. coli grown on sucrose and hence (along with thechloramphenicol marker and pSC101 origin) is used to force and selectfor de-integration and plasmid curing events. This methodology had beendescribed previously (Hamilton et al. 4617-22; Blomfield et al.1447-57). The pKO3 system removes all selective markers from the hostgenome except for the inserted gene.

The Following Plasmid was Constructed:

pMXE191 comprising the knockout mutated Tsp gene as shown in the SEQ IDNO: 28 comprising EcoR I and Ase I restriction markers.

The plasmid was then transformed into electro-competent E. coli W3110cells prepared using the method found in Miller and Nickoloff, which isincorporated herein by reference in its entirety (Miller and Nickoloff105-13).

Day 1: 40 μl of E. coli cells were mixed with (10 pg) 1 μl of pKO3 DNAin a chilled BioRad 0.2 cm electroporation cuvette beforeelectroporation at 2500V, 25 μF and 200Ω. 1000 μl of 2xPY was addedimmediately, the cells recovered by shaking at 250 rpm in an incubatorat 30° C. for 1 hour. Cells were serially 1/10 diluted in 2xPY before100 μl aliquots were plated out onto 2xPY agar plates containingchloramphenicol at 20 μg/ml prewarmed at 30° C. and 43° C. Plates wereincubated overnight at 30° C. and 43° C.

Day 2: The number of colonies grown at 30° C. gave an estimate of theefficiency of electroporation, while colonies that survive growth at 43°C. represent potential integration events. Single colonies from the 43°C. plate were picked and resuspended in 10 ml of 2xPY. 100 μl of thiswas plated out onto 2xPY agar plates containing 5% (w/v) sucrosepre-warmed to 30° C. to generate single colonies. Plates were incubatedovernight at 30° C.

Day 3: Colonies here represent potential simultaneous de-integration andplasmid curing events. If the de-integration and curing events happenedearly on in the growth, then the bulk of the colony mass will be clonal.Single colonies were picked and replica plated onto 2xPY agar thatcontained either chloramphenicol at 20 μg/ml or 5% (w/v) sucrose. Plateswere incubated overnight at 30° C.

Day 4: Colonies that both grow on sucrose and die on chloramphenicolrepresent potential chromosomal replacement and plasmid curing events.These were picked and screened by PCR with a mutation-specificoligonucleotide. Colonies that generated a positive PCR band of thecorrect size were struck out to produce single colonies on 2xPY agarcontaining 5% (w/v) sucrose and the plates were incubated overnight at30° C.

Day 5: Single colonies of PCR-positive, chloramphenicol-sensitive andsucrose-resistant E. coli were used to make glycerol stocks andchemically competent cells and act as PCR templates for a PCR reactionwith 5′ and 3′ flanking oligonucleotide primers to generate PCR productfor direct DNA sequencing using Taq polymerase.

Cell strain MXE001 was tested to confirm successful modification ofgenomic DNA carrying the mutated Tsp gene by PCR amplification of theregion of the Tsp gene comprising a non-naturally occurring Ase Irestriction site (SEQ ID NO: 28) using oligonucleotide primers. Theamplified regions of the DNA were then analyzed by gel electrophoresisbefore and after incubation with Ase I restriction enzyme to confirm thepresence of the non-naturally occurring Ase I restriction site in themutated genes. This method was carried out as follows:

The following oligos were used to amplify, using PCR, genomic DNA fromprepared E. coli cell lysates from MXE001 and W3110:

6284 Tsp 3′ (SEQ ID NO: 47) 5′-GCATCATAATTTTCTTTTTACCTC-3′ 6283 Tsp 5′(SEQ ID NO: 48) 5′-GGGAAATGAACCTGAGCAAAACGC-3′

The lysates were prepared by heating a single colony of cells for 10minutes at 95° C. in 20 μl of 1×PCR buffer. The mixture was allowed tocool to room temperature, then centrifuged at 13,200 rpm for 10 minutes.The supernatant was removed and labeled as “cell lysate”.

Each strain was amplified using the Tsp oligonucleotide pair.

The DNA was amplified using a standard PCR procedure.

5 μl Buffer ×10 (Roche) 1 μl dNTP mix (Roche, 10 mM mix) 1.5 μl 5′ oligo(5 pmol) 1.5 μl 3′ oligo (5 pmol) 2 μl Cell lysate 0.5 μl Taq DNApolymerase (Roche 5 U/μl) 38.5 μl H₂O PCR cycle. 94° C.  1 minute 94° C. 1 minute 55° C.  1 minute (repeated for 30 cycles) 72° C.  1 minute 72°C. 10 minutes

Once the reactions were complete 25 μl was removed to a new microfugetube for digestion with Ase I. To the 25 μl of PCR reaction 19 μl ofH₂O, 5 μl of Buffer 3 (New England Biolabs®), and 1 μl of Ase I (NewEngland Biolabs®) were added, mixed and incubated at 37° C. for 2 hours.

To the remaining PCR reaction 5 μl of loading buffer (×6) was added and20 μl was loaded onto a 0.8% TAE 200 ml agarose gel (Invitrogen®) plusethidium bromide (5 μl of 10 mg/ml stock) and run at 100 V for 1 hour.10 μl of size marker (Perfect DNA marker 0.1-12 Kb, Novagen®) was loadedin the final lane.

Once the Ase I digestions were complete 10 μl of of loading buffer (×6)was added and 20 μl was loaded onto a 0.8% TAE agarose gel (Invitrogen®)plus ethidium bromide (5 μl of 10 mg/ml stock) and run at 100 V for 1hour. 10 μl of size marker (Perfect DNA marker 0.1-12 Kb, Novagen®) wasloaded in the final lane. Both gels were visualized using a UVtransluminator.

The genomic fragment amplified showed the correct sized band of 2.8 Kbfor Tsp. Following digestion with Ase I this confirmed the presence ofthe introduced Ase I sites in the Tsp-deficient strain MXE001 but not inthe W3110 control.

MXE001: genomic DNA was amplified using the Tsp primer set and theresulting DNA was digested with Ase I to produce 2.2 and 0.6 Kbps bands.

W3110 PCR amplified DNA was not digested by Ase I restriction enzyme.

Example 2—Generation of Cell Lines Carrying Mutated Spr Gene and CellLines Carrying a Mutated Tsp Gene and a Mutated Spr Gene

The spr mutations were generated and selected for using acomplementation assay.

The spr gene (SEQ ID NO: 23) was mutated using the Clontech® randommutagenesis diversity PCR kit which introduced 1 to 2 mutations per 1000bp. The mutated spr PCR DNA was cloned into an inducible expressionvector [pTTO CDP870] which expresses CDP870 Fab′ (as described in WO01/94585) along with the spr mutant. This ligation was thenelectro-transformed into an E. coli strain comprising a deletion variantof Tsp (ΔTsp) (designated MXE001) prepared using the method found inMiller et al. (Miller and Nickoloff 105-13). The following protocol wasused: 40 μl of electro-competent MXE001, 2.5 μl of the ligation (100 pgof DNA) was added to a 0.2 cm electroporation cuvette, andelectro-transformation was performed using as BioRad® Gene Pulser Xcell®with the following conditions: 2500 V, 25 μF and 200Ω. After theelectro-transformation 1 ml of S.O.C. medium (Invitrogen® catalog:18045-088) (pre-warmed to 37° C.) was added and the cells left torecover at 37° C. for 1 hour with gentle agitation.

The cells were plated onto hypotonic agar [5 g/L yeast extract, 2.5 g/Ltryptone, 15 g/L agar (all Difco®)] and incubated at 40° C. Cells whichformed colonies were re-plated onto HLB at 43° C. to confirm restorationof the ability to grow under low osmotic conditions at high temperatureto the MXE001 strain. Plasmid DNA was prepared from the selected clonesand sequenced to identify spr mutations.

Using this method eight single, one double and two multiple mutations inthe spr protein were isolated which complemented the ΔTsp phenotype asfollows:

1. V98E

2. D133A

3. V135D

4. V135G

5. G147C

6. S95F and Y115F

7. I70T

8. N31T, Q73R, R100G, and G140C

9. R62C, Q99P, and R144C

10. L108S

11. L136P.

The individual mutations 1 to 5 identified above and three catalytictriad mutations of spr (C94A, H145A, H157A) and W174R were used togenerate new strains using either the wild-type W3110 E. coli strain(genotype: F-LAM-IN (rrnD-rrnE)1 rph1 (ATCC catalog no. 27325)) tocreate spr mutated strains or the MXE001 strain from Example 1 to makecombined ΔTsp/mutant spr strains.

The following mutant E. coli cell strains were generated using a genereplacement vector system using the pKO3 homologousrecombination/replacement plasmid (Link et al., supra) as described inExample 1 for the generation of MXE001.

TABLE 1 Mutant E. coli Strain Genotype Spr Vectors MXE001 ΔTsp — MXE008ΔTsp, spr D133A pMXE339, pK03 spr D133A (-SalI) MXE009 ΔTsp, spr H157ApMXE345, pK03 spr H157A (-SalI) MXE010 spr G147C pMXE338, pK03 spr G147C(-SalI) MXE011 spr C94A pMXE343, pK03 spr C94A (-SalI) MXE012 spr H145ApMXE344, pK03 spr H145A (-SalI) MXE013 spr W174R pMXE346, pK03 spr W174R(-SalI) MXE014 ΔTsp, spr V135D pMXE340, pK03 spr V135D (-SalI) MXE015ΔTsp, spr V98E pMXE342, pK03 spr V98E (-SalI) MXE016 ΔTsp, spr C94ApMXE343, pK03 spr C94A (-SalI) MXE017 ΔTsp, spr H145A pMXE344, pK03 sprH145A (-SalI) MXE018 ΔTsp, spr V135G pMXE341, pK03 spr V135G (-SalI)

The mutant spr integration cassettes were moved as Sal I, Not Irestriction fragments into similarly restricted pKO3 plasmids.

For all experiments the E. coli cell line W3110 was used as thewild-type cell line and the E. coli cell line W3110 ΔTsp, spr C94A(MX016).

Example 3—Generation of Plasmid for Anti-CD154 Fab′ and DsbC Expression

A plasmid was constructed containing both the heavy and light chainsequences of an anti-CD154 Fab (SEQ ID NOs: 13 and 11, respectively) andthe sequence encoding DsbC (SEQ ID NO: 27).

Plasmid pMXE351 (pTTOD_DsbC), an expression vector for the anti-CD154Fab and DsbC (a periplasmic polypeptide), was constructed usingconventional restriction cloning methodologies. The plasmid pMXE351contained the following features: a strong tac promoter and lac operatorsequence. As shown in FIG. 1, the plasmid contained a unique EcoRIrestriction site after the coding region of the Fab′ heavy chain,followed by a non-coding sequence and then a unique NdeI restrictionsite. The DsbC gene was PCR cloned using W3110 crude chromosomal DNA asa template. An EcoRI site was removed from the wild-type DsbC sequenceby PCR overlap extension such that the PCR product encoded for a 5′EcoRI site followed by a strong ribosome binding site, followed by thenative start codon, signal sequence and mature sequence of DsbC,terminating in a C-terminal His tag and finally a non-coding NdeI site.The EcoRI-NdeI PCR fragment was restricted and ligated into theexpression vector such that all three polypeptides (Fab′ light chain,Fab′ heavy chain and DsbC) were encoded on a single polycistronic mRNA.

The Fab light chain and heavy chain genes and DsbC gene were transcribedas a single polycistronic message. DNA encoding the signal peptide fromthe E. coli OmpA protein was fused to the 5′ end of both light and heavychain gene sequences, which directed the translocation of thepolypeptides to the E. coli periplasm. Transcription was terminatedusing a dual transcription terminator rrnB tlt2. The lacIq gene encodedthe constitutively expressed Lac I repressor protein. This repressedtranscription from the tac promoter until de-repression was induced bythe presence of allolactose or IPTG. The origin of replication used wasp15A, which maintained a low copy number. The plasmid contained atetracycline resistance gene for antibiotic selection.

Example 4—Expression of Anti-CD154 Fab′ and DsbC in E. Coli W3110 andMXE016 (E. Coli W3110 ΔTsp, Spr C94A)

Expression of Anti-CD154 Fab′ and DsbC in E. Coli W3110 ΔTsp, Spr C94A

The E. coli W3110 ΔTsp, spr C94A cell strain (MXE016) was transformedwith the plasmid pMXE351 generated in Example 3. The transformation ofthe strains was carried out using the method found in Chung, C. T. etal. (Chung, Niemela, and Miller 2172-75).

Expression of Anti-CD154 Fab′ in E. Coli W3110

The E. coli W3110 cell strain was transformed with plasmid pTTOD, anexpression vector for the anti-CD154 Fab′, which was constructed usingconventional restriction cloning methodologies. The plasmid pTTODcontained the following features: a strong tac promoter and lac operatorsequence. The Fab light and heavy chain genes were transcribed as asingle dicistronic message. DNA encoding the signal peptide from the E.coli OmpA protein was fused to the 5′ end of both light and heavy chaingene sequences, which directed the translocation of the polypeptides tothe E. coli periplasm. Transcription was terminated using a dualtranscription terminator rrnB tlt2. The lacIq gene encoded theconstitutively expressed Lac I repressor protein. This repressedtranscription from the tac promoter until de-repression was induced bythe presence of allolactose or IPTG. The origin of replication used wasp15A, which maintained a low copy number. The plasmid contained atetracycline resistance gene for antibiotic selection. Thetransformation of the strains was carried out using the method found inChung, C. T. et al. (Chung, Niemela, and Miller 2172-75).

Example 5—Growth of Mutated E. Coli Strains and Expression of Anti-CD154Fab′ in Mutated E. Coli Strains Using High Density Fermentations

The strains produced in Example 4 were tested in fermentationexperiments comparing growth and expression of an anti-CD154 Fab′.

Growth Medium, Inoculum and Fermentation Steps.

The fermentation process is initiated by preparing an inoculum from avial of the cell bank and amplifying through several pre-culture stages(flask and reactors) before seeding of the production fermenter. In theproduction fermenter, the cells are grown in defined media to highdensity in batch and fed-batch mode. When the desired cell density isreached expression of the Fab′ is induced by the addition of IPTG. TheFab′ expression is targeted to the E. coli periplasmic space, where Fab′accumulates throughout the course of the induction phase. A carbonsource feed is applied during the induction phase to control expressionand cell growth. Temperature, dissolved oxygen (pO₂) and pH arecontrolled to maintain the culture within optimal culture conditions.

Measurement of Biomass Concentration and Growth Rate.

Biomass concentration was determined by measuring the optical density ofcultures at 600 nm.

Periplasmic Extraction.

Cells were collected from culture samples by centrifugation. Thesupernatant fraction was retained (at −20° C.) for further analysis. Thecell pellet fraction was resuspended to the original culture volume inextraction buffer (100 mM Tris-HCl, 10 mM EDTA; pH 7.4). Followingincubation at 60° C. for approximately 10 to 16 hours the extract wasclarified by centrifugation and the supernatant fraction used fresh orretained (at −20° C.) for analysis.

Fab′ Quantification.

Fab′ concentrations in periplasmic extracts and culture supernatantswere determined by using Protein G HPLC. A HiTrap® Protein-G HP 1 mlcolumn (GE Healthcare® or equivalent) was loaded with analyte(approximately neutral pH, 30° C., 0.2 μm filtered) at 2 ml/min, thecolumn was washed with 20 mM phosphate, 50 mM NaCl, pH 7.4 and then Fab′was eluted using an injection of 50 mM glycine/HCl, pH 2.7. Eluted Fab′was measured by A280 on an Agilent® 1100 or 1200 HPLC system andquantified by reference to a standard curve of a purified Fab′ proteinof known concentration.

Example 6—Level of Light Chain Fragments in Fermentation Extractions ofMutated E. Coli Strains

The fermentations presented in Example 2 were tested for light chainfragment level of anti-CD154 Fab′ in periplasmic extractions.

Light Chain Fragment Quantification.

Quantitative estimation of the level of Fab′ and Fab′ proteolyticfragments was achieved by high-temperature reversed phase HPLC.Separation is performed on a Poroshell® 300SB-C8 reversed phase column(Agilent Technologies®, Product No. 660750-906) at a temperature of 80°C. The equilibration solvent is HPLC water, 0.1% (v/v) TFA, and theelution solvent is 80:20 (v/v) 1-propanol:acetonitrile, 0.03% (v/v) TFA.Separation is performed at a flow rate of 2.0 mL/min, by means of alinear gradient of 16-38% solvent B in 4.4 min. Detection was by UVabsorbance at 214 nm. Data were processed by manual integration, and thequantity of Fab proteolytic fragments expressed as % peak area relativeto the intact Fab peak.

The present invention also provides a therapeutic or diagnosticcomposition comprising the antibody produced by the method of thepresent invention in combination with a pharmaceutically acceptableexcipient, diluent or carrier.

The present invention also provides a process for preparation of atherapeutic or diagnostic composition comprising admixing the antibodyproduced by the method of the present invention together with apharmaceutically acceptable excipient, diluent or carrier.

While this invention has been particularly shown and described withreference to preferred embodiments, it will be understood to thoseskilled in the art that various changes in form and detail may be madewithout departing from the scope of the invention as defined by theappending claims.

REFERENCE LIST

-   Aramini, J. M., et al. “Solution NMR structure of the N1pC/P60    domain of lipoprotein Spr from Escherichia coli: structural evidence    for a novel cysteine peptidase catalytic triad.” Biochemistry. 47.37    (2008): 9715-17.-   Bachmann, B. J. “Pedigrees of some mutant strains of Escherichia    coli K-12.” Bacteriol. Rev. 36.4 (1972): 525-57.-   Backlund, E., et al. “Fedbatch design for periplasmic product    retention in Escherichia coli.” J. Biotechnol. 135.4 (2008): 358-65.-   Blomfield, I. C., et al. “Allelic exchange in Escherichia coli using    the Bacillus subtilis sacB gene and a temperature-sensitive pSC101    replicon.” Mol. Microbiol. 5.6 (1991): 1447-57.-   Chung, C. T., S. L. Niemela, and R. H. Miller. “One-step preparation    of competent Escherichia coli: transformation and storage of    bacterial cells in the same solution.” Proc. Natl. Acad. Sci. U.S.A.    86.7 (1989): 2172-75.-   Hamilton, C. M., et al. “New method for generating deletions and    gene replacements in Escherichia coli.” J. Bacteriol. 171.9 (1989):    4617-22.-   Hara, H., et al. “Overproduction of penicillin-binding protein 7    suppresses thermosensitive growth defect at low osmolarity due to an    spr mutation of Escherichia coli.” Microb. Drug Resist. 2.1 (1996):    63-72.-   Hara, H., et al. “Cloning, mapping, and characterization of the    Escherichia coli prc gene, which is involved in C-terminal    processing of penicillin-binding protein 3.” J. Bacteriol. 173.15    (1991): 4799-813.-   Holliger, P. and P. J. Hudson. “Engineered antibody fragments and    the rise of single domains.” Nat. Biotechnol. 23.9 (2005): 1126-36.-   Humphreys, D. P., et al. “Formation of dimeric Fabs in Escherichia    coli: effect of hinge size and isotype, presence of interchain    disulphide bond, Fab′ expression levels, tail piece sequences and    growth conditions.” J. Immunol. Methods. 209.2 (1997): 193-202.-   Keiler, K. C. and R. T. Sauer. “Identification of active site    residues of the Tsp protease.” J. Biol. Chem. 270.48 (1995):    28864-68.-   Link, A. J., D. Phillips, and G. M. Church. “Methods for generating    precise deletions and insertions in the genome of wild-type    Escherichia coli: application to open reading frame    characterization.” J. Bacteriol. 179.20 (1997): 6228-37.-   Makrides, S. C. “Strategies for achieving high-level expression of    genes in Escherichia coli.” Microbiol. Rev. 60.3 (1996): 512-38.-   Miller, E. M. and J. A. Nickoloff. “Escherichia coli    electrotransformation.” Methods Mol. Biol. 47:105-13. (1995):    105-13.-   Missiakas, D., C. Georgopoulos, and S. Raina. “The Escherichia coli    dsbC (xprA) gene encodes a periplasmic protein involved in disulfide    bond formation.” EMBO J. 13.8 (1994): 2013-20.-   Nagasawa, H., et al. “Determination of the cleavage site involved in    C-terminal processing of penicillin-binding protein 3 of Escherichia    coli.” J. Bacteriol. 171.11 (1989): 5890-93.-   Shevchik, V. E., G. Condemine, and J. Robert-Baudouy.    “Characterization of DsbC, a periplasmic protein of Erwinia    chrysanthemi and Escherichia coli with disulfide isomerase    activity.” EMBO J. 13.8 (1994): 2007-12.-   Silber, K. R., K. C. Keiler, and R. T. Sauer. “Tsp: a tail-specific    protease that selectively degrades proteins with nonpolar C    termini.” Proc. Natl. Acad. Sci. U.S.A. 89.1 (1992): 295-99.-   Silber, K. R. and R. T. Sauer. “Deletion of the prc (tsp) gene    provides evidence for additional tail-specific proteolytic activity    in Escherichia coli K-12.” Mol. Gen. Genet. 242.2 (1994): 237-40.

We claim:
 1. A recombinant gram-negative bacterial cell comprising thefollowing genetic modifications: (a) an expression vector comprising arecombinant polynucleotide encoding a protein comprising DsbC and ahistidine-tag, said histidine tag being at the N-terminus or C-terminusof DsbC; (b) a polynucleotide encoding a reduced periplasmic Prcprotease (Tsp) activity compared to a wild-type cell; (c) one or morepolynucleotides encoding an antibody or an antigen binding fragmentthereof specifically binding to CD154; and (d) optionally, apolynucleotide encoding a mutant extragenic suppressor (spr) protein,wherein the cell is isogenic to a wild-type bacterial cell except forthe genetic modifications (a) to (c) and when present, (d).
 2. Thegram-negative bacterial cell according to claim 1, wherein theexpression vector comprises a polynucleotide having the sequence givenin SEQ ID NO: 45 or SEQ ID NO:
 51. 3. The gram-negative bacterial cellaccording to claim 1, wherein the antibody or antigen binding fragmentthereof comprises a heavy chain variable domain comprising three CDRshaving the sequence given in SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2, forCDRH2 and SEQ ID NO: 3 for CDRH3 and a variable domain light chaincomprising three CDRs having the sequence given in SEQ ID NO: 4 forCDRL1, SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3.
 4. Thegram-negative bacterial cell according to claim 3, wherein the one ormore polynucleotides encode an antibody comprising the light chainvariable region sequence given in SEQ ID NO: 8 and the heavy chainvariable region given in SEQ ID NO:
 10. 5. The gram-negative bacterialcell according to claim 1, wherein the antibody is a Fab or Fab′fragment.
 6. The gram-negative bacterial cell according to claim 5,wherein the Fab or Fab′ fragment comprises a light chain having thesequence given in SEQ ID NO: 12 and a heavy chain having the sequencegiven in SEQ ID NO: 14 or
 16. 7. The gram-negative bacterial cellaccording to claim 1, wherein the gram-negative bacterial cell comprisesa first expression vector comprising the recombinant polynucleotideencoding DsbC and a second expression vector comprising one or morepolynucleotides encoding the antibody or an antigen binding fragmentthereof specifically binding to CD154.
 8. The gram-negative bacterialcell according to claim 1, wherein the expression vector comprising therecombinant polynucleotide encoding DsbC additionally comprises one ormore polynucleotides encoding an antibody or an antigen binding fragmentthereof specifically binding to CD154.
 9. The gram-negative bacterialcell according to claim 1, wherein the cell comprises a recombinantpolynucleotide encoding the mutant spr protein.
 10. A recombinantgram-negative bacterial cell comprising the following geneticmodifications: an expression vector comprising a recombinantpolynucleotide encoding DsbC; nucleic acid modifications that reduceperiplasmic Prc protease (Tsp) activity as compared to a wild-type cell;one or more polynucleotides encoding an antibody or an antigen bindingfragment thereof specifically binding to CD154; and a polynucleotideencoding a mutated extragenic suppressor (spr) protein wherein thepolynucleotide encoding the mutant spr protein comprises a mutationaffecting the amino acid C94 and, wherein the cell comprises a knock-outmutation of the polynucleotide encoding Tsp protein.
 11. Thegram-negative bacterial cell according to claim 10, wherein the mutationin the polynucleotide encoding the mutant spr protein results in achange of the amino acid cysteine to alanine at position 94 (C94A). 12.The gram-negative bacterial cell according to claim 1, wherein the cellis E. coli.
 13. The gram-negative bacterial cell according to claim 1,wherein the cell comprises an expression vector comprising therecombinant polynucleotide encoding DsbC and a dicistronic message forproducing the antibody or antigen binding fragment thereof specificallybinding to CD154, in which the upstream cistron contains DNA coding forthe light chain of the antibody and the downstream cistron contains DNAcoding for the corresponding heavy chain, characterised in that thedicistronic message comprises a sequence selected from IGS1 (SEQ ID NO:33), IGS2 (SEQ ID NO: 34), IGS3 (SEQ ID NO: 35) and IGS4 (SEQ ID NO:36).
 14. An expression vector, which comprises a recombinantpolynucleotide encoding a protein comprising DsbC and a histidine tag atthe N-terminus or the C-terminus and a dicistronic message for producingan antibody or antigen binding fragment thereof specifically binding toCD154, in which the upstream cistron contains DNA coding for the lightchain of the antibody and the downstream cistron contains DNA coding forthe corresponding heavy chain, characterised in that the dicistronicmessage comprises a sequence selected from IGS1 (SEQ ID NO: 33), IGS2(SEQ ID NO: 34), IGS3 (SEQ ID NO: 35) and IGS4 (SEQ ID NO: 36).
 15. Theexpression vector according to claim 14, wherein the antibody or anantigen binding fragment thereof specifically binding to CD154 comprisesa heavy chain variable domain comprising three CDRs having the sequencegiven in SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2, for CDRH2 and SEQ ID NO:3 for CDRH3 and a variable domain light chain comprising three CDRshaving the sequence given in SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 forCDRL2 and SEQ ID NO: 6 for CDRL3.
 16. A method for producing an antibodyor an antigen binding fragment thereof specifically binding to CD154comprising: (a) culturing a recombinant gram-negative bacterial cellaccording to claim 1 in a culture medium under conditions effective toexpress the antibody or the antigen binding fragment thereofspecifically binding to CD154 and the recombinant polynucleotideencoding DsbC; and (b) recovering the antibody or an antigen bindingfragment thereof specifically binding to CD154 from the periplasm of therecombinant gram-negative bacterial cell and/or the culture medium. 17.The method according to claim 16, wherein the method further comprises astep of attaching an effector molecule to an amino acid at or towardsthe C-terminal end of the heavy chain and/or the light chain of theantibody.
 18. The method according to claim 17, wherein the effectormolecule comprises poly(ethyleneglycol) or methoxypoly(ethyleneglycol).19. The method according to claim 18, wherein the method comprisesattaching to one of the cysteine residues at the C-terminal end of theheavy chain a lysyl-maleimide group wherein each amino group of thelysyl residue has covalently linked to it a methoxypoly(ethyleneglycol)residue having a molecular weight of about 20,000 Da.
 20. A method forproducing an antibody or an antigen binding fragment thereofspecifically binding to CD154 comprising: (a) culturing a recombinantgram-negative bacterial cell according to claim 10 in a culture mediumunder conditions effective to express the antibody or the antigenbinding fragment thereof specifically binding to CD154 and therecombinant polynucleotide encoding DsbC; and (b) recovering theantibody or an antigen binding fragment thereof specifically binding toCD154 from the periplasm of the recombinant gram-negative bacterial celland/or the culture medium.